乌头碱对大鼠心肌细胞毒性作用的分子毒理学机制研究
|
摘要
1研究背景
有毒动植物中毒是具有中国特色法医毒理学的重要组成部分，也是我国法医毒理学研究的重点内容之一。乌头与乌头属植物是我国临床常用的重要中药，是药用有毒植物的典型代表，也为我国最早有记载的有毒植物，其主要毒性成分为乌头类生物碱，其中以含有双酯基化学结构的乌头碱毒性最强。乌头碱的主要毒性作用的靶器官，主要为心脏与神经系统。有大量研究表明，乌头碱的毒性成份，也是其药性成份，具有强心、镇痛、抗炎、抗肿瘤、降低血压、降低血管通透性等作用，广泛地被应用于临床治疗。由于乌头碱的毒性剧烈，其有效治疗剂量与中毒剂量或致死量极为接近，用药稍有不慎，如因炮制不当，或误服等，即可引起中毒甚至死亡。而利用乌头属植物及乌头碱自杀、投毒他杀的案件时有发生，可见乌头碱中毒在有毒动植物中毒中占有重要位置。
为了进一步提高乌头碱中毒的诊断、治疗，规范乌头属有毒中药在疾病中的使用，同时，也为乌头碱中毒的法医学鉴定提供相关的理论依据，对乌头碱中毒机制研究，特别是乌头碱对心肌细胞的毒性作用机制研究，成为了中药研究与应用、临床急救医学和法医毒理学研究的重点问题。
近年来，国内外对乌头碱心肌细胞毒性作用机制的研究，已进入到细胞质膜动态变化的分子作用机理水平。但大多仍局限于心肌细胞损伤及单个心肌细胞离子通道检测等方面，对毒物靶器官心室肌细胞群毒性作用的分子毒理学机制，特别是乌头碱对心肌细胞之间信息传递的影响机制、基因表达调控机制尚不明确。
2研究目的
2．1系统观察不同浓度乌头碱染毒后心肌细胞的毒理病理学变化，研究不同中毒剂量、中毒时间与病变间的效应关系，优化、规范乌头碱靶器官的细胞培养及分子毒理学研究的基础方法；
2．2研究乌头碱中毒与心肌细胞DNA损伤的关系，为从分子毒理学角度研究DNA损伤与乌头碱中毒机制的关系提供基础；
2．3研究乌头碱染毒对心肌细胞Ca~(2+)依赖性蛋白激酶Cα亚型(PKCα)的表达、PKCα磷酸化水平，以及PKCα磷酸化对Cx43磷酸化表达的影响，明确其内在级联效应关系，及参与乌头碱心肌细胞毒性作用分子机制的途径；
2．4研究乌头碱对心肌细胞内Ca~(2+)调控蛋白表达的影响，观察Ca~(2+)调控蛋白参与乌头碱心肌细胞毒性作用机制的方式；
2．5研究乌头碱中毒特征及法医学鉴定注意事项。
3研究方法
3．1新生大鼠心肌细胞原代培养及其方法优化研究
选择1～2d的SD新生大白鼠，雄雌不限，消毒后直接剪取心室肌，用胰蛋白酶等消化液消化，制作心肌细胞悬液；于37℃、5％CO_2培养箱，以差速贴壁法培养原代心肌细胞，优化心肌细胞原代培养方法，提高心室肌细胞纯度、成活率和心肌细胞群整体性。选用台盼蓝染色法进行培养心肌细胞活性鉴定；采用抗心肌特异性单克隆抗体，结合间接免疫荧光法检测，对心肌细胞纯度进行鉴定。规范、优化新生大鼠心肌细胞体外原代培养的方法，为后续研究建立良好地基础。
3．2乌头碱染毒模型的建立及毒性效应研究
于心肌细胞原代培养第6天，更换无血清培养液培养16～18h，以去除血清干扰，再加入不同浓度乌头碱，构建心肌细胞乌头碱染毒模型，根据实验目的不同，设定不同的阳性和阴性对照。实时动态监测乌头碱染毒心肌细胞的形态、功能的变化，研究乌头碱的毒性对心肌细胞损伤的量效作用。
3．3乌头碱对大鼠心肌培养细胞DNA损伤的研究
新生SD大鼠24只，随机分为6组；取心室肌以差速贴壁法原代培养心室肌细胞。培养第6天，更换无血清培养液培养后，制成密度为2×10~5个细胞／mL的细胞悬液，分别加入浓度为0.1、0.5、1.0、2.0μmol／L的乌头碱混合液，染毒30 min；并以PBS溶液作阴性对照。采用彗星电泳技术及CASP分析软件，检测不同浓度乌头碱染毒后心室肌细胞DNA损伤程度。实验重复6次。
3．4乌头碱对新生大鼠心肌培养细胞内PKCα表达的影响
取12孔原代心肌培养细胞组，分成对照组(Normal)、乌头碱染毒组(ACO)、碱性磷酸酶处理组(A．P．)、AAP干预组(AAP)、AAP干预后乌头碱染毒组(AAP+ACO)、PKCα抑制剂G(o|¨) 6976干预组(G(o|¨) 6976)，及AAP与G(o|¨) 6976联合干预组(AAP+G(o|¨) 6976)，共7组。提取各实验组及对照组心肌细胞总蛋白，以BCA法标准蛋白曲线定量，采用Western-blotting技术，选用P-Cx43(Ser-368)蛋白抗体、NP-Cx43(Ser-368)蛋白抗体、抗PKCα蛋白抗体，及P-PKCα(Ser-657)蛋白抗体，定量检测各组在培养心肌细胞内P-Cx43(Ser-368)蛋白、NP-Cx43(Ser-368)蛋白、总PKCα蛋白，及P-PKCα(Ser-657)蛋白表达含量变化。实验重复6次。
3．5乌头碱对心肌细胞PKCα及P-PKCα(Ser-657)位点磷酸化状态的影响
选择兔抗鼠总PKCα多克隆抗体(稀释度为1：200)、兔抗鼠磷酸化PKCα(Ser-657)(稀释度为1：200)功能性抗体，用Fluorescein标记山羊抗兔IgG和Rhodamine标记山羊抗兔IgG(稀释度为1：100)，应用激光共聚焦扫描显微镜，结合细胞图像荧光定分析技术，检测乌头碱染毒前后，心肌细胞激酶Cα亚型及其第657位丝氨酸残基(Ser657)位点，特异性磷酸化状态的改变。
3．6乌头碱对大鼠心肌培养细胞Ca~(2+)调控蛋白表达的影响
取12孔原代心肌培养细胞组，运用荧光标记技术及RT-PCR技术，建立钠钙交换体(NCX)、肌浆网钙泵Ca~(2+)-ATP酶(SERCA_2)、磷酸受钠蛋白(PLB)、兰尼碱受体(RyR_2)，及管家基因β-actin共5个基因座的多重荧光复合RT-PCR反应体系及扩增方法，在3100 DNA测序仪中进行毛细管电泳，检测乌头碱染毒组及正常对照组心肌细胞内NCX、SERCA_2、RyR_2、PLB四种基因mRNA表达的差异与变化。实验重复6次。
3．7乌头碱中毒10例法医学尸检资料分析
收集10例乌头碱中毒死亡的法医学鉴定资料，包括系统的法医学解剖资料及组织病理学检查结果，部分案例还有案情调查、抢救病历资料等；并按年龄、性别、中毒原因、中毒类型、病理变化及特征等分类整理。结乌头碱中毒的毒理病理组织学变化，探讨乌头碱中毒法医学鉴定的注意事项。
3．8实验数据的统计学分析
本实验研究采用SSPS统计软件(version 10.0，USA)分析，数据以(?)±s表示。
3．8．1彗星电泳结果采用CASP软件分析HDNA％、TDNA％、TL、TM、OTM，5个检测指标。计算各组平均值和标准差，以(?)±s表示，应用SSPS在方差齐性条件下做单因素方差分析，对各剂量组组间差异进行比较分析。
3．8．2正常对照组和各实验组中P-Cx43(Ser-368)蛋白表达和NP-Cx43(Ser-368)蛋白表达差异、总PKCα蛋白表达和P-PKCα(Ser-657)蛋白表达相对含量分析，应用SSPS做单变量两因素方差分析(ANOVA)，对组间均数的多重比较选用Games-Howell检验。
3．8．3对ca~(2+)调控蛋白NCX、SERCA_2、RyR_2、PLB基因mRNA表达的差异与变化，应用SSPS做t检验。
4研究结果
4．1原代培养心肌细胞及其方法优化
在倒置相差显微镜下观察，刚接种时的心肌细胞悬浮于培养液中，为均一、分散的圆形折光颗粒；12 h后细胞已开始贴壁生长，偶尔可见个别细胞自发搏动；培养24 h后，贴壁细胞互相连接呈稀疏网状，并出现缓慢的同步化搏动；48 h后，心肌细胞在培养孔底部进一步伸展，呈现快速同步化搏动的细胞单层，搏动频率约80～120次／min；培养第6天，心肌细胞在培养孔底部充分伸展，形成稳定的同步化搏动细胞单层，频率约120次／min。采用台盼蓝染色法测定，活细胞的存活率平均达到97.33％；选用抗心肌肌钙蛋白Ⅰ(cTnⅠ)特异性单克隆抗体，结合免疫荧光法检测，心肌培养细胞平均细胞纯度为97.1％。
4．2乌头碱对心肌培养细胞染毒模型的建立及毒性效应观察
建立乌头碱染毒模型，与正常对照组比较，不同剂量乌头碱染毒后，心肌细胞出现非同步性搏动，搏动减慢，甚至停博。心肌细胞乌头碱染毒后，毒理病理变化与中毒剂量、中毒时间呈现一定的相关性。
4．3乌头碱对大鼠心肌培养细胞DNA损伤的观察
心肌培养细胞被不同浓度乌头碱染毒后，尾部DNA含量、彗尾长度、尾矩、Olive-尾矩均随乌头碱浓度增加而升高，头部DNA含量则逐渐降低，与对照组相比，均有极显著性差异(P＜0.01)；结果显示，乌头碱染毒剂量越大，心肌细胞DNA损伤越严重。
4．4乌头碱对心肌培养细胞内PKCα蛋白表达的影响
4．4．1对心肌细胞内P-Cx43(Ser-368)表达的观察
各实验组均以Normal为参照。不同干预组对P-Cx43(Ser-368)蛋白表达均有所不同。与正常对照组比较，ACO、A．P．、G(o|¨) 6976组心肌细细胞P-Cx43(Ser-368)蛋白表达均下降；AAP、AAP+ACO、AAP+G(o|¨)9676组P-Cx43(Ser-368)蛋白表达均上升。选用Games-Howell统计方法，检验多组间均数的多重比较，显示ACO、A．P．、G(o|¨) 6976三组间，仅ACO染毒组具有统计学差异(P＜0.05)；而AAP、AAP+ACO、AAP+G(o|¨)9676三个干预组之间未见明显差异。从Western Blotting检测结果显示：ACO、A．P．、G(o|¨) 6976干预后，Cx43(Ser-368)位点磷酸化蛋白表达减少；经APP预处理后，磷酸化表达增强，且效果明显(P＜0.01)。
4．4．2对心肌细胞内NP-Cx43(Ser-368)表达的观察
各组NP-Cx43(Ser-368)位点磷酸化状态蛋白定量分析结果显示：与正常对照比较，A．P．组、ACO染毒组、G(o|¨) 6976组表达增强(P＜0.01)，其中A．P．组表达最强。而AAP组、AAP+ACO组、AAP+G(o|¨) 6976联用组则表达降低(P＜0.01)。
4．4．3对心肌细胞中总PKCα表达的观察
各组总PKCα蛋白定量分析结果显示：与正常对照比较，A．P．组、ACO染毒组、G(o|¨) 6976组、AAP组、AAP+ACO组、AAP+G(o|¨) 6976联用组心肌细胞内总PKCα表达水平无显著差异(P＞0.05)。
4．4．4对心肌细胞中总P-PKCα(Ser-657)表达的观察
各实验组均以Normal为参照。不同实验干预组P-PKCα(Ser-657)蛋白均有不同程度的表达。正常组和ACO组心肌细胞内均有P-PKCα(Ser-657)蛋白表达，而A．P．组心肌细胞内无P-PKCα(Ser-657)蛋白表达。
与正常对照组相比较，ACO组、G(o|¨) 6976组心肌细胞中P—PKCα(Ser-657)磷酸化蛋白表达显著降低(P＜0.01)。而AAP组、AAP+ACO组、AAP+G(o|¨) 6976联合处理组，磷酸化表达增强，心肌细胞P-PKCα(Ser-657)蛋白表达显著增高(P＜0.01)。
4．4．5乌头碱染毒前后心肌细胞PKCα及PKCα(Ser-657)位点磷酸化状态的观察应用激光扫描共聚焦显微镜，观察免疫荧光检测结果显示：
标记总PKCα的Fluorescein呈绿色荧光信号，弥散性分布于心肌细胞胞浆中。与正常对照组相比较，ACO染毒组、G(o|¨) 6976组、AAP组、AAP+ACO组、AAP+G(o|¨) 6976联合处理组总PKCα绿色荧光信号差异无显著性。
标记P-PKCα(Ser-657)的Rhodamine呈红色荧光信号，弥散性分布于心肌细胞胞浆中。与正常对照组相比较，ACO染毒组、G(o|¨) 6976组，红色荧光信号显著减弱，有极显著性差异(P＜0.01)；而AAP组、AAP+ACO组、AAP+G(o|¨) 6976联合处理组红色荧光信号较正常对照组，也有极显著性差异(P＜0.01)。
4．5乌头碱对大鼠心肌培养细胞Ca~(2+)调控蛋白表达的影响
多重荧光复合RT-PCR检测发现，与正常对照组比较，乌头碱染毒组RyR_2、NCX基因mRNA表达增加，而PLB、SERCA_2基因mRNA表达减少。
4．6乌头碱中毒尸检资料分析
病理变化特征主要为：
①心肌灶性出血，细胞横纹不清，肌浆凝聚，细胞间质淤血；
②肝可见肝细胞灶性或点状坏死，可见肝细胞脂肪变性或水样变性；
③肺脏可有灶性或点状出血，灶性肺水肿；
④可见部分胃粘膜有散在性点状出血；
⑤偶见肾组织点状出血和散在性坏死。
5研究结论
5．1优化、规范心肌细胞培养方法，建立稳定的体外新生大鼠培养细胞的乌头碱染毒模式，为法医毒理学研究提供了方法学上的参考。
5．2乌头碱染毒可引起心肌细胞DNA损伤，并呈明显的剂量—效应关系，推测细胞DNA损伤参与乌头碱毒性作用机制。
5．3乌头碱染毒可影响PKCα本身的磷酸化状态，同时乌头碱染毒所致的PKCα(Ser657)位点蛋白磷酸化表达下降，可进一步导致心肌细胞Cx43磷酸化状态减弱。推测PKCα磷酸化、Cx43磷酸化状态的改变是乌头碱心肌细胞毒性作用机制之一。
5．4乌头碱可影响心肌细胞Ca~(2+)调控蛋白的表达，使RyR_2、NCX基因mRNA表达增加，PLB、SERCA_2基因mRNA表达减少，推测Ca~(2+)调控蛋白参与了乌头碱毒性作用机制。其毒性作用机制的方式仍有待进一步研究。。Ca~(2+)调控蛋白参与毒性作用机制有待进一步研究。
5．5对10例乌头碱中毒尸检资料进行分析，总结其毒理病理变化特点，并提出乌头碱中毒法医学鉴定的注意事项。
1 Background
Toxic animals and plants poisoning are one of important componentelements of forensic toxicology with Chinese characteristic, and a part ofimportant content of forensic toxicology research in China. Aconite andAconitium plants, which are important Chinese medicine widely used inclinic, are typical representative for herb poisonous plants, and they arethe poisonous plant recorded earliest. Aconitium alkaloid is their chiefingredient; and Aconitine, chemical structure of which containsdiesterditerpene, is the most poisonous. The target organs of aconitinetoxic effect are mainly heart and nervous system. It has been confirmedthat toxic ingredient of aconitine, which plays roles of drug, have widespread pharmaceutical properties including cardio-tonic, analgesic,anti-inflammatory, tumor-suppressant, lowering blood pressure, reducinghemal wall and so on. As aconitine are of severe toxicity and therapy doseis so close to poisonous dose or lethal dose that use immodestly, such asimproper process, misuse and so on, would lead to poisoning, even death.Meanwhile, related homicide or veneficium suicide using Aconite andAconitium plants are matters of common occurrences, which indicate thatAconitine intoxication lies important position of toxic animals and plantspoisoning.
In order to further improve the diagnosis and healing of aconitinepoisoning, specify the therapeutic action of Aconitium poisonous Chinesetraditional medicines for diseases and offer relevant theoretical foundationfor forensic evaluation of aconitine intoxication, the investigation of the mechanism of aconitine poisoning, especially the mechanism of toxiceffect on cardiomyocytes, turn into the important issue for research andapplication of Chinese traditional medicines, clinical emergency medicineand forensic toxicology study.
At present, mechanism studies of toxic effect on aconitine poisoningare mainly focused on molecular action of cytoplasma membrane dynamicchange, most of which are limited to cardiomyocytes injury, detection ofion channel of single cardiac myocyte and so on. It is uncertain thatmolecular toxic mechanism of toxic effect on groups of ventricularmyocytes as target organ, especially influence on information transferbetween cardiomyocytes and mechanism of gene expression regulatory.
2 Objectives
●To systematic observe toxicological pathology change of cardiomyocytesafter incubation with different concentrations of aconitine,investigate the effect relationship between different poisonous dose,poisonous period and pathological change, optimize and specify cell ofaconitine target organ culture and basic method of moleculartoxicologic study.
●To study the DNA damage of aconitine cultured cardiomyocytes andoffer theoretical principle for the relationship between DNA damageand aconitine intoxication mechanism from molecular toxicologicpoint of view.
●To investigate the expression of protein kinase C (PKCα) andphosphorylation level in aconitine cultured cardiomyocytes and howPKCαphosphorylation affect Cx43 (Ser368) phosphorylation; affirmwhether PKCαand P-Cx43 (Ser368) phosphorylation be of cascadeeffect relation after aconitine incubation and play role in molecularmechanism of aconitine cardiomyocytes.
●To explore the influence of aconitine on calmodulin in cardiomyocytes.Observe calmodulinin involved in the way of toxicitymechanism of aconitine cardiomyocytes.
●To analyze the character of aconitine poisoning and attentions inforensic identification.
3 Methods
3.1 Optimizing neonatal rats' cardomyocytes primary culturecondition.
1-2 day-old Spague-Dawley rats, regardless of the gender, weredisinfected, and ventricles were minced and digested in solutioncontaining trypsinase to be myocardial cell suspension. After incubation at37℃in 5% CO_2, selective attachment of cardiomyocytes was induced forprimary culture cell to optimize primary cardomyocytes culture conditionand increase the purity, survival rates of ventricular myocytes and integrityof cardomyocytes group. Identify the activity of cultured cardiomyocytesby trypan blue staining method, and evaluate the purity of cardiomyocytesby anti-myocardium specific monoclonal antibody combining withindirect immunofluorescence. Specify and optimize in vitro neonatal rats'cardomyocytes primary culture condition and set up preferred basis forfollowing study.
3.2 Setting up aconitine cultured model and studying toxic effect ofaconitine.
On 6~(th) day of primary cardiomyocytes culture, after replenishmentwith serum-free medium for 16～18h incubation to wipe off interference ofserum, cardiomyocytes was incubated with different doses of aconitine tobe cultured models. Meanwhile, set up various intoxicated and controlgroups according to different objectives. Real-time monitoringmorphologic and functional change of aconitine cultured cardiomyocytes, and analyze the dose-effect manner of aconitine toxicity tocardiomyocytes injury.
3.3 Studying the DNA damage of aconitine cultured cardiomyoeytes.
Ventricular myocytes of 24 neonatal rats (randomly divided into 6groups) were incubated by differential adherent culture. On 6~(th) day ofcardiomyocytes primary culture, after replenishment with serum-freemedium for 16～18h incubation, unattached cells were adjusted to cellsuspension at a density of 2×10~5 cell/ml. Add aconitine solution in cellsuspensions to aconitine mixture at different concentrations of 0.1μM/L,0.5μM/L, 1μM/L and 2μM/L for 30 min incubation. Set PBS to benegative control group. Detect DNA damage level of ventricular myocytesafter aconitine incubation by comet assay and acridine orange dye andanalyze the damage by CASP analysis software. All the steps repeated 6times.
3.4 Investigating the expression of PKCαin aconitine culturedneonatal rats' cardiomyocytes.
12 wells for primary cultured cardiomyocytes group were dividedinto 7groups, including normal group (Normal), aconitine incubated group(ACO), alkaline phosphatase treated group (A.P.), AAP treated group(AAP), aconitine incubated after AAP treated group (AAP+ACO), G(o|¨)6976 group (PKCαdepressant, G(o|¨) 6976) and AAP and G(o|¨) 6976 co-treatedgroup (AAP+G(o|¨) 6976). Extract total protein of cardiomyocytes ofexperimental groups and control group, and quantify the total protein byBCA standard protein regression curve. Quantify the change of expressionamounts of P-Cx43 (Ser-368), NP-Cx43 (Ser-368) and total PKCαandP-PKCα(Ser-657) in groups of cultured cardiomyocytes byWestern-bloting with anti-P-Cx43 (Ser-368) antibody, anti-NP-Cx43(Ser-368) antibody and anti-PKCαantibody and anti-P-PKCα(Ser-657)antibody. All the steps repeated 6 times.
3.5 Influence of the PKCαand P-PKCα(Ser-657) locusphosphorylation state in cardiomyocytes after aconitine incubation.
Anti-rabbit-anti-rat PKCαpolyclonal antibody (dilution at 1:200),rabbit anti-mouse phosphorylation PKCα(Ser-657) functional antibody(dilution at 1:200), Fluorescein mark goat via rabbit IgG and Rhodaminemark goat via rabbit IgG (dilution at 1:100) were selected to detect thespecific phosphorylation state of P-PKCα(Ser-657) locus with laserscanning confocal microscope and cellular image fluorescent locationanalysis technique before and after aconitine incubation.
3.6 Influence of the expression of calmodulin in aconitine culturedrats' cardiomyocytes.
Multiplex fluorescence reaction system and amplification methodwere set up with Na~+-Ca~(2+) crossover (NCX), phosphoric receptor sodiumalbumen (PLB), sarcoplasmic reticulum calcium pump Ca~(2+)-ATP ase(SERCA_2), Ryanodine receptor (RyR_2) and housekeeping gene (β-actin)by fluorescence labeling technique and RT-PCR in 12 wells of primarycultured cardiomyocytes groups. Capillary electrophoresis was used by3100 DNA Sequencer to detect the difference and change of transcriptmRNA of NCX, SERCA_2, RyR_2 and PLB in aconitine cultured groups andcontrol group. All the steps repeated 6 times.
3.7 Analyzing forensic postmortem examination documents of 10aconitine poisoning cases.
Collect forensic postmortem examination documents of 10 aconitinepoisoning cases, including systematic forensic anatomical data andhistopathological outcomes, investigation documents, rescue record and soon for some of the cases. Sort the data by age, gender, poisoning cause,poisoning type, and pathological change, characteristic and so on.Summarize the toxicological pathologic change of aconitine poisoning and investigate attention of forensic identification for aconitine poisoning.
3.8 Statistical analysis of experimental data.
Data from the study analyzed by SSPS statistical software (version10.0, USA) were expressed as (?)±s.
●Outcomes of comet assay were processed by CASP analysis software.Experimental data, such as head DNA (HDNA %), tail DNA(TDNA %), tail length (TL), tail moment (TM) and Olive tail moment(OTM) were measured and average value and standard deviation werecalculated and expressed by (?)±s. Each dosage group was comparedby one-way analysis of variance (ANOVA) with homogeneity ofvariance, and differences between groups were analyzed.
●The differences between expressions of P-Cx43 (Ser-368) andNP-Cx43 (Ser-368), relative expression amounts of total PKCαandP-PKCα(Ser-657) from control group and each experimental groupwere analyzed with univariate two-way (ANOVA) by SSPS statisticalsoftware; and multiple comparisons were made using Games-Howellpost hoc test.
●The difference and change of transcript mRNA of NCX, SERCA_2,RyR_2 and PLB in aconitine cultured groups and control group wereanalyzed by t-test with SPSS statistical software.
4 Results
4.1 Primary cardiomyocytes culture and condition optimization.
When plated in culture well, the cardiomyocytes were bright pellets inshape and suspended in medium. After 12 hours, they began to crawl onthe bottom of the culture well and spontaneous beating could be found infew cells. After 24 hours, attached myocytes connected each other, looked like a net; and slow synchronous beating could be found in all cultures.After 48 hours, the cardiomyocytes stretched on the bottom of culturewells, and high-speed (80～120 times per minute) synchronous beatingmonolayers of syncytium were formed. On 6th day of culture, meanlivability of cardiomyocytes were 97.33 % using trypan blue stainingmethod. Applying immunofluorescent microscopy with specificmonoclonal anti-cTnI antibody, we estimated that the mean purity ofcultured cardiomyocytes were 97.1%.
4.2 Setting-up models of cultured cardiomyocytes treated by aconitineand observing the toxic effect.
Aconitine incubated models were set up and it was verified by H.E.staining that the morphological structure of cardiomyocytes were hardlychanged after incubation with different dosages of aconitine compared tothe control cultures. But cardiomyocytes showed asynchronic beat,slowing beat and even arrest. There were certain correlation amongtoxicological pathologic change, toxic dose and poisoning period.
4.3 Observation of DNA damage of cultured rats' cardiomyocytesafter aconitine incubation.
After different concentrations of aconitine incubation, tail DNA, taillength, tail moment ~(TM) and Olive tail moment of cultured neonatal rats'cardiomyocytes increased, and head DNA decreased while theconcentration of aconitine increased. And there were all extremelysignificant differences compared with the control group (P＜0.01). Theoutcomes indicated that higher the dose of aconitine, severer the DNAdamage of cardiomyocytes.
4.4 The influence of expression of PKCαin cultured cardiomyocytes.
4.4.1 Observation of expression of P-Cx43 (Ser-368) in cardimyocytes.
Every experimental group was compared with the normal group. Andthe expressions of P-Cx43 (Ser-368) in cardiomyocytes with different treated patterns were not the same. The expression amounts of P-Cx43(Ser-368) in cardiomyocytes of ACO incubated group, A.R group and G(o|¨)6976 group decreased, and all increased in AAP group, AAP+ACO groupand AAP+G(o|¨) 6976 group. Games-Howell statistical method was selectedfor multiple comparisons of means between groups. Among ACOincubated group, A.P. group and G(o|¨) 6976 group, there was significantstatistical difference in ACO incubated group (P＜0.05), while there werehardly obvious difference among AAP group, AAP+ACO group andAAP+G(o|¨) 6976 group. The outcomes by Western Blotting presented thatafter ACO, A.R and G(o|¨) 6976 treated, the expression protein of Cx43(Ser-368) locus decreased, while after APP pre-treated increased obviously(P＜0.01).
4.4.2 Observation of expression of NP-Cx43(Ser-368) in cardiomyocytes.
The quantitative analytic outcomes of expression protein of NP-Cx43(Ser-368) locus phosphorylation state presented that the expression in A.P.group, ACO incubated group and G(o|¨) 6976 group reinforce (P＜0.01),especially A.P. group the most, while AAP group, AAP+ACO group andAAP+G(o|¨) 6976 group reduce (P＜0.01).
4.4.3 Observation of expression of total PKCαin cardiomyocytes.
The quantitative analytic result of total PKCαshowed that there wereno significant difference of expression of total PKCαin A.R group, ACOincubated group, G(o|¨) 6976 group, AAP group, AAP+ACO group andAAP+G(o|¨) 6976 group, compared with the control group (P＞0.05).
4.4.4 Observation of expression of total P- PKCα(Ser-657) incardiomyocytes.
Every experimental group was compared with the normal group. Andthe expressions of P-PKCα(Ser-657) in cardiomyocytes with differenttreated patterns were different. The P-PKCα(Ser-657) expressed incardiomyocytes of the normal group and ACO group, but none of A.Rgroup.
Compared with the normal group, the expressions of P-PKCα (Ser-657) phosphorylation protein in cardiomyocytes of ACO group andG(o|¨) 6976 group decreased obviously (P＜0.01). And the expressions ofphosphorylation increased in AAP group, AAP+ACO group and AAP+G(o|¨)6976 group (P＜0.01).
4.4.5 Observation of PKCαand PKCα(Ser-657) locus phosphorylationstate before and after aconitine incubation
Applying laser scanning confocal microscope, the immumofluorescenedetection results presented:
Total PKCαlabeled by Fluorescein, as green fluorescene signals, weredispersed distributed in cytoplasm of cardiomyocytes. There were nosignificant differences in ACO incubated group, G(o|¨) 6976 group, AAPgroup, AAP+ACO group and AAP+G(o|¨) 6976 co-treated group comparedwith the normal group.
Total P-PKCα(Ser-657) labeled by Rhodamine red fluorescene signalslabeling, as red fluorescene signals, were dispersed distributed incytoplasm of cardiomyocytes. The red fluorescene signals weakenedobviously and there were extremely significant differences in ACOincubated group and G(o|¨) 6976 group compared with the normal group(P＜0.01). There were also extremely significant differences of redfluorescene signals in AAP group, AAP+ACO group and AAP+G6 6976co-treated group (P＜0.01).
4.5 Influence of expression of calmodulin in aconitine cultured rats'cardiomyocytes.
It was detected that mRNA of RyR_2 and NCX expressed more, andPLB and SERCA_2 less by RT-PCR combined with multiplex fluorescence.
4.6 Analysis of forensic postmortem examination documents ofaconitine poisoning cases.
Characteristic of pathological changes were mainly that:①myocardium focal hemorrhage, transverse striation of cardiomyocytes wasunclear, sarcoplasm condensed and intercellular congestion;②focal or spotty necrosis of hepatocytes and fatty degeneration or hydropicdegeneration;③focal or globular hemorrhage of lung, focalpneumonedema;④scattered globular hemorrhage in gastric mucosa ofsome cases;⑤globular hemorrhage or scattered necrosis in kidney of afew cases.
5 Conclusions
●5.1 Optimization and specify cardiomyocytes culture method,establishment of aconitine incubated in vitro neonatal rats' cell modeland reference for forensic toxicologic study.
●5.2 DNA damage of aconitine incubated cardiomyocytes was ofobvious dose-effect relation, which would indicate that DNA damageplayed a role in toxic effect mechanism.
●5.3 Aconitine incubation would influence phosphorylation state ofPKCαand phosphorylation expression of PKCα(Ser657) decreased,which further induced that phosphorylated Cx43 of cardiomyocytesweakened. It inferred that the change of phosphorylation state ofPKCαand Cx43 was one of the paths of the aconitine poisoning.
●5.4 Aconitine influenced the expression of calmodulin incardiomyocytes, and mRNA of RyR_2 and NCX expressed more andPLB and SERCA_2 less. That calmodulin take part in toxic effectmechanism deserves further research.
●5.5 Announcements of forensic identification for aconitine poisoningwere raised after collection of forensic postmortem examinationdocuments of 10 aconitine poisoning cases and summarize thecharacters of toxicological pathologic change.
有毒动植物中毒是具有中国特色法医毒理学的重要组成部分，也是我国法医毒理学研究的重点内容之一。乌头与乌头属植物是我国临床常用的重要中药，是药用有毒植物的典型代表，也为我国最早有记载的有毒植物，其主要毒性成分为乌头类生物碱，其中以含有双酯基化学结构的乌头碱毒性最强。乌头碱的主要毒性作用的靶器官，主要为心脏与神经系统。有大量研究表明，乌头碱的毒性成份，也是其药性成份，具有强心、镇痛、抗炎、抗肿瘤、降低血压、降低血管通透性等作用，广泛地被应用于临床治疗。由于乌头碱的毒性剧烈，其有效治疗剂量与中毒剂量或致死量极为接近，用药稍有不慎，如因炮制不当，或误服等，即可引起中毒甚至死亡。而利用乌头属植物及乌头碱自杀、投毒他杀的案件时有发生，可见乌头碱中毒在有毒动植物中毒中占有重要位置。
为了进一步提高乌头碱中毒的诊断、治疗，规范乌头属有毒中药在疾病中的使用，同时，也为乌头碱中毒的法医学鉴定提供相关的理论依据，对乌头碱中毒机制研究，特别是乌头碱对心肌细胞的毒性作用机制研究，成为了中药研究与应用、临床急救医学和法医毒理学研究的重点问题。
近年来，国内外对乌头碱心肌细胞毒性作用机制的研究，已进入到细胞质膜动态变化的分子作用机理水平。但大多仍局限于心肌细胞损伤及单个心肌细胞离子通道检测等方面，对毒物靶器官心室肌细胞群毒性作用的分子毒理学机制，特别是乌头碱对心肌细胞之间信息传递的影响机制、基因表达调控机制尚不明确。
2研究目的
2．1系统观察不同浓度乌头碱染毒后心肌细胞的毒理病理学变化，研究不同中毒剂量、中毒时间与病变间的效应关系，优化、规范乌头碱靶器官的细胞培养及分子毒理学研究的基础方法；
2．2研究乌头碱中毒与心肌细胞DNA损伤的关系，为从分子毒理学角度研究DNA损伤与乌头碱中毒机制的关系提供基础；
2．3研究乌头碱染毒对心肌细胞Ca~(2+)依赖性蛋白激酶Cα亚型(PKCα)的表达、PKCα磷酸化水平，以及PKCα磷酸化对Cx43磷酸化表达的影响，明确其内在级联效应关系，及参与乌头碱心肌细胞毒性作用分子机制的途径；
2．4研究乌头碱对心肌细胞内Ca~(2+)调控蛋白表达的影响，观察Ca~(2+)调控蛋白参与乌头碱心肌细胞毒性作用机制的方式；
2．5研究乌头碱中毒特征及法医学鉴定注意事项。
3研究方法
3．1新生大鼠心肌细胞原代培养及其方法优化研究
选择1～2d的SD新生大白鼠，雄雌不限，消毒后直接剪取心室肌，用胰蛋白酶等消化液消化，制作心肌细胞悬液；于37℃、5％CO_2培养箱，以差速贴壁法培养原代心肌细胞，优化心肌细胞原代培养方法，提高心室肌细胞纯度、成活率和心肌细胞群整体性。选用台盼蓝染色法进行培养心肌细胞活性鉴定；采用抗心肌特异性单克隆抗体，结合间接免疫荧光法检测，对心肌细胞纯度进行鉴定。规范、优化新生大鼠心肌细胞体外原代培养的方法，为后续研究建立良好地基础。
3．2乌头碱染毒模型的建立及毒性效应研究
于心肌细胞原代培养第6天，更换无血清培养液培养16～18h，以去除血清干扰，再加入不同浓度乌头碱，构建心肌细胞乌头碱染毒模型，根据实验目的不同，设定不同的阳性和阴性对照。实时动态监测乌头碱染毒心肌细胞的形态、功能的变化，研究乌头碱的毒性对心肌细胞损伤的量效作用。
3．3乌头碱对大鼠心肌培养细胞DNA损伤的研究
新生SD大鼠24只，随机分为6组；取心室肌以差速贴壁法原代培养心室肌细胞。培养第6天，更换无血清培养液培养后，制成密度为2×10~5个细胞／mL的细胞悬液，分别加入浓度为0.1、0.5、1.0、2.0μmol／L的乌头碱混合液，染毒30 min；并以PBS溶液作阴性对照。采用彗星电泳技术及CASP分析软件，检测不同浓度乌头碱染毒后心室肌细胞DNA损伤程度。实验重复6次。
3．4乌头碱对新生大鼠心肌培养细胞内PKCα表达的影响
取12孔原代心肌培养细胞组，分成对照组(Normal)、乌头碱染毒组(ACO)、碱性磷酸酶处理组(A．P．)、AAP干预组(AAP)、AAP干预后乌头碱染毒组(AAP+ACO)、PKCα抑制剂G(o|¨) 6976干预组(G(o|¨) 6976)，及AAP与G(o|¨) 6976联合干预组(AAP+G(o|¨) 6976)，共7组。提取各实验组及对照组心肌细胞总蛋白，以BCA法标准蛋白曲线定量，采用Western-blotting技术，选用P-Cx43(Ser-368)蛋白抗体、NP-Cx43(Ser-368)蛋白抗体、抗PKCα蛋白抗体，及P-PKCα(Ser-657)蛋白抗体，定量检测各组在培养心肌细胞内P-Cx43(Ser-368)蛋白、NP-Cx43(Ser-368)蛋白、总PKCα蛋白，及P-PKCα(Ser-657)蛋白表达含量变化。实验重复6次。
3．5乌头碱对心肌细胞PKCα及P-PKCα(Ser-657)位点磷酸化状态的影响
选择兔抗鼠总PKCα多克隆抗体(稀释度为1：200)、兔抗鼠磷酸化PKCα(Ser-657)(稀释度为1：200)功能性抗体，用Fluorescein标记山羊抗兔IgG和Rhodamine标记山羊抗兔IgG(稀释度为1：100)，应用激光共聚焦扫描显微镜，结合细胞图像荧光定分析技术，检测乌头碱染毒前后，心肌细胞激酶Cα亚型及其第657位丝氨酸残基(Ser657)位点，特异性磷酸化状态的改变。
3．6乌头碱对大鼠心肌培养细胞Ca~(2+)调控蛋白表达的影响
取12孔原代心肌培养细胞组，运用荧光标记技术及RT-PCR技术，建立钠钙交换体(NCX)、肌浆网钙泵Ca~(2+)-ATP酶(SERCA_2)、磷酸受钠蛋白(PLB)、兰尼碱受体(RyR_2)，及管家基因β-actin共5个基因座的多重荧光复合RT-PCR反应体系及扩增方法，在3100 DNA测序仪中进行毛细管电泳，检测乌头碱染毒组及正常对照组心肌细胞内NCX、SERCA_2、RyR_2、PLB四种基因mRNA表达的差异与变化。实验重复6次。
3．7乌头碱中毒10例法医学尸检资料分析
收集10例乌头碱中毒死亡的法医学鉴定资料，包括系统的法医学解剖资料及组织病理学检查结果，部分案例还有案情调查、抢救病历资料等；并按年龄、性别、中毒原因、中毒类型、病理变化及特征等分类整理。结乌头碱中毒的毒理病理组织学变化，探讨乌头碱中毒法医学鉴定的注意事项。
3．8实验数据的统计学分析
本实验研究采用SSPS统计软件(version 10.0，USA)分析，数据以(?)±s表示。
3．8．1彗星电泳结果采用CASP软件分析HDNA％、TDNA％、TL、TM、OTM，5个检测指标。计算各组平均值和标准差，以(?)±s表示，应用SSPS在方差齐性条件下做单因素方差分析，对各剂量组组间差异进行比较分析。
3．8．2正常对照组和各实验组中P-Cx43(Ser-368)蛋白表达和NP-Cx43(Ser-368)蛋白表达差异、总PKCα蛋白表达和P-PKCα(Ser-657)蛋白表达相对含量分析，应用SSPS做单变量两因素方差分析(ANOVA)，对组间均数的多重比较选用Games-Howell检验。
3．8．3对ca~(2+)调控蛋白NCX、SERCA_2、RyR_2、PLB基因mRNA表达的差异与变化，应用SSPS做t检验。
4研究结果
4．1原代培养心肌细胞及其方法优化
在倒置相差显微镜下观察，刚接种时的心肌细胞悬浮于培养液中，为均一、分散的圆形折光颗粒；12 h后细胞已开始贴壁生长，偶尔可见个别细胞自发搏动；培养24 h后，贴壁细胞互相连接呈稀疏网状，并出现缓慢的同步化搏动；48 h后，心肌细胞在培养孔底部进一步伸展，呈现快速同步化搏动的细胞单层，搏动频率约80～120次／min；培养第6天，心肌细胞在培养孔底部充分伸展，形成稳定的同步化搏动细胞单层，频率约120次／min。采用台盼蓝染色法测定，活细胞的存活率平均达到97.33％；选用抗心肌肌钙蛋白Ⅰ(cTnⅠ)特异性单克隆抗体，结合免疫荧光法检测，心肌培养细胞平均细胞纯度为97.1％。
4．2乌头碱对心肌培养细胞染毒模型的建立及毒性效应观察
建立乌头碱染毒模型，与正常对照组比较，不同剂量乌头碱染毒后，心肌细胞出现非同步性搏动，搏动减慢，甚至停博。心肌细胞乌头碱染毒后，毒理病理变化与中毒剂量、中毒时间呈现一定的相关性。
4．3乌头碱对大鼠心肌培养细胞DNA损伤的观察
心肌培养细胞被不同浓度乌头碱染毒后，尾部DNA含量、彗尾长度、尾矩、Olive-尾矩均随乌头碱浓度增加而升高，头部DNA含量则逐渐降低，与对照组相比，均有极显著性差异(P＜0.01)；结果显示，乌头碱染毒剂量越大，心肌细胞DNA损伤越严重。
4．4乌头碱对心肌培养细胞内PKCα蛋白表达的影响
4．4．1对心肌细胞内P-Cx43(Ser-368)表达的观察
各实验组均以Normal为参照。不同干预组对P-Cx43(Ser-368)蛋白表达均有所不同。与正常对照组比较，ACO、A．P．、G(o|¨) 6976组心肌细细胞P-Cx43(Ser-368)蛋白表达均下降；AAP、AAP+ACO、AAP+G(o|¨)9676组P-Cx43(Ser-368)蛋白表达均上升。选用Games-Howell统计方法，检验多组间均数的多重比较，显示ACO、A．P．、G(o|¨) 6976三组间，仅ACO染毒组具有统计学差异(P＜0.05)；而AAP、AAP+ACO、AAP+G(o|¨)9676三个干预组之间未见明显差异。从Western Blotting检测结果显示：ACO、A．P．、G(o|¨) 6976干预后，Cx43(Ser-368)位点磷酸化蛋白表达减少；经APP预处理后，磷酸化表达增强，且效果明显(P＜0.01)。
4．4．2对心肌细胞内NP-Cx43(Ser-368)表达的观察
各组NP-Cx43(Ser-368)位点磷酸化状态蛋白定量分析结果显示：与正常对照比较，A．P．组、ACO染毒组、G(o|¨) 6976组表达增强(P＜0.01)，其中A．P．组表达最强。而AAP组、AAP+ACO组、AAP+G(o|¨) 6976联用组则表达降低(P＜0.01)。
4．4．3对心肌细胞中总PKCα表达的观察
各组总PKCα蛋白定量分析结果显示：与正常对照比较，A．P．组、ACO染毒组、G(o|¨) 6976组、AAP组、AAP+ACO组、AAP+G(o|¨) 6976联用组心肌细胞内总PKCα表达水平无显著差异(P＞0.05)。
4．4．4对心肌细胞中总P-PKCα(Ser-657)表达的观察
各实验组均以Normal为参照。不同实验干预组P-PKCα(Ser-657)蛋白均有不同程度的表达。正常组和ACO组心肌细胞内均有P-PKCα(Ser-657)蛋白表达，而A．P．组心肌细胞内无P-PKCα(Ser-657)蛋白表达。
与正常对照组相比较，ACO组、G(o|¨) 6976组心肌细胞中P—PKCα(Ser-657)磷酸化蛋白表达显著降低(P＜0.01)。而AAP组、AAP+ACO组、AAP+G(o|¨) 6976联合处理组，磷酸化表达增强，心肌细胞P-PKCα(Ser-657)蛋白表达显著增高(P＜0.01)。
4．4．5乌头碱染毒前后心肌细胞PKCα及PKCα(Ser-657)位点磷酸化状态的观察应用激光扫描共聚焦显微镜，观察免疫荧光检测结果显示：
标记总PKCα的Fluorescein呈绿色荧光信号，弥散性分布于心肌细胞胞浆中。与正常对照组相比较，ACO染毒组、G(o|¨) 6976组、AAP组、AAP+ACO组、AAP+G(o|¨) 6976联合处理组总PKCα绿色荧光信号差异无显著性。
标记P-PKCα(Ser-657)的Rhodamine呈红色荧光信号，弥散性分布于心肌细胞胞浆中。与正常对照组相比较，ACO染毒组、G(o|¨) 6976组，红色荧光信号显著减弱，有极显著性差异(P＜0.01)；而AAP组、AAP+ACO组、AAP+G(o|¨) 6976联合处理组红色荧光信号较正常对照组，也有极显著性差异(P＜0.01)。
4．5乌头碱对大鼠心肌培养细胞Ca~(2+)调控蛋白表达的影响
多重荧光复合RT-PCR检测发现，与正常对照组比较，乌头碱染毒组RyR_2、NCX基因mRNA表达增加，而PLB、SERCA_2基因mRNA表达减少。
4．6乌头碱中毒尸检资料分析
病理变化特征主要为：
①心肌灶性出血，细胞横纹不清，肌浆凝聚，细胞间质淤血；
②肝可见肝细胞灶性或点状坏死，可见肝细胞脂肪变性或水样变性；
③肺脏可有灶性或点状出血，灶性肺水肿；
④可见部分胃粘膜有散在性点状出血；
⑤偶见肾组织点状出血和散在性坏死。
5研究结论
5．1优化、规范心肌细胞培养方法，建立稳定的体外新生大鼠培养细胞的乌头碱染毒模式，为法医毒理学研究提供了方法学上的参考。
5．2乌头碱染毒可引起心肌细胞DNA损伤，并呈明显的剂量—效应关系，推测细胞DNA损伤参与乌头碱毒性作用机制。
5．3乌头碱染毒可影响PKCα本身的磷酸化状态，同时乌头碱染毒所致的PKCα(Ser657)位点蛋白磷酸化表达下降，可进一步导致心肌细胞Cx43磷酸化状态减弱。推测PKCα磷酸化、Cx43磷酸化状态的改变是乌头碱心肌细胞毒性作用机制之一。
5．4乌头碱可影响心肌细胞Ca~(2+)调控蛋白的表达，使RyR_2、NCX基因mRNA表达增加，PLB、SERCA_2基因mRNA表达减少，推测Ca~(2+)调控蛋白参与了乌头碱毒性作用机制。其毒性作用机制的方式仍有待进一步研究。。Ca~(2+)调控蛋白参与毒性作用机制有待进一步研究。
5．5对10例乌头碱中毒尸检资料进行分析，总结其毒理病理变化特点，并提出乌头碱中毒法医学鉴定的注意事项。
1 Background
Toxic animals and plants poisoning are one of important componentelements of forensic toxicology with Chinese characteristic, and a part ofimportant content of forensic toxicology research in China. Aconite andAconitium plants, which are important Chinese medicine widely used inclinic, are typical representative for herb poisonous plants, and they arethe poisonous plant recorded earliest. Aconitium alkaloid is their chiefingredient; and Aconitine, chemical structure of which containsdiesterditerpene, is the most poisonous. The target organs of aconitinetoxic effect are mainly heart and nervous system. It has been confirmedthat toxic ingredient of aconitine, which plays roles of drug, have widespread pharmaceutical properties including cardio-tonic, analgesic,anti-inflammatory, tumor-suppressant, lowering blood pressure, reducinghemal wall and so on. As aconitine are of severe toxicity and therapy doseis so close to poisonous dose or lethal dose that use immodestly, such asimproper process, misuse and so on, would lead to poisoning, even death.Meanwhile, related homicide or veneficium suicide using Aconite andAconitium plants are matters of common occurrences, which indicate thatAconitine intoxication lies important position of toxic animals and plantspoisoning.
In order to further improve the diagnosis and healing of aconitinepoisoning, specify the therapeutic action of Aconitium poisonous Chinesetraditional medicines for diseases and offer relevant theoretical foundationfor forensic evaluation of aconitine intoxication, the investigation of the mechanism of aconitine poisoning, especially the mechanism of toxiceffect on cardiomyocytes, turn into the important issue for research andapplication of Chinese traditional medicines, clinical emergency medicineand forensic toxicology study.
At present, mechanism studies of toxic effect on aconitine poisoningare mainly focused on molecular action of cytoplasma membrane dynamicchange, most of which are limited to cardiomyocytes injury, detection ofion channel of single cardiac myocyte and so on. It is uncertain thatmolecular toxic mechanism of toxic effect on groups of ventricularmyocytes as target organ, especially influence on information transferbetween cardiomyocytes and mechanism of gene expression regulatory.
2 Objectives
●To systematic observe toxicological pathology change of cardiomyocytesafter incubation with different concentrations of aconitine,investigate the effect relationship between different poisonous dose,poisonous period and pathological change, optimize and specify cell ofaconitine target organ culture and basic method of moleculartoxicologic study.
●To study the DNA damage of aconitine cultured cardiomyocytes andoffer theoretical principle for the relationship between DNA damageand aconitine intoxication mechanism from molecular toxicologicpoint of view.
●To investigate the expression of protein kinase C (PKCα) andphosphorylation level in aconitine cultured cardiomyocytes and howPKCαphosphorylation affect Cx43 (Ser368) phosphorylation; affirmwhether PKCαand P-Cx43 (Ser368) phosphorylation be of cascadeeffect relation after aconitine incubation and play role in molecularmechanism of aconitine cardiomyocytes.
●To explore the influence of aconitine on calmodulin in cardiomyocytes.Observe calmodulinin involved in the way of toxicitymechanism of aconitine cardiomyocytes.
●To analyze the character of aconitine poisoning and attentions inforensic identification.
3 Methods
3.1 Optimizing neonatal rats' cardomyocytes primary culturecondition.
1-2 day-old Spague-Dawley rats, regardless of the gender, weredisinfected, and ventricles were minced and digested in solutioncontaining trypsinase to be myocardial cell suspension. After incubation at37℃in 5% CO_2, selective attachment of cardiomyocytes was induced forprimary culture cell to optimize primary cardomyocytes culture conditionand increase the purity, survival rates of ventricular myocytes and integrityof cardomyocytes group. Identify the activity of cultured cardiomyocytesby trypan blue staining method, and evaluate the purity of cardiomyocytesby anti-myocardium specific monoclonal antibody combining withindirect immunofluorescence. Specify and optimize in vitro neonatal rats'cardomyocytes primary culture condition and set up preferred basis forfollowing study.
3.2 Setting up aconitine cultured model and studying toxic effect ofaconitine.
On 6~(th) day of primary cardiomyocytes culture, after replenishmentwith serum-free medium for 16～18h incubation to wipe off interference ofserum, cardiomyocytes was incubated with different doses of aconitine tobe cultured models. Meanwhile, set up various intoxicated and controlgroups according to different objectives. Real-time monitoringmorphologic and functional change of aconitine cultured cardiomyocytes, and analyze the dose-effect manner of aconitine toxicity tocardiomyocytes injury.
3.3 Studying the DNA damage of aconitine cultured cardiomyoeytes.
Ventricular myocytes of 24 neonatal rats (randomly divided into 6groups) were incubated by differential adherent culture. On 6~(th) day ofcardiomyocytes primary culture, after replenishment with serum-freemedium for 16～18h incubation, unattached cells were adjusted to cellsuspension at a density of 2×10~5 cell/ml. Add aconitine solution in cellsuspensions to aconitine mixture at different concentrations of 0.1μM/L,0.5μM/L, 1μM/L and 2μM/L for 30 min incubation. Set PBS to benegative control group. Detect DNA damage level of ventricular myocytesafter aconitine incubation by comet assay and acridine orange dye andanalyze the damage by CASP analysis software. All the steps repeated 6times.
3.4 Investigating the expression of PKCαin aconitine culturedneonatal rats' cardiomyocytes.
12 wells for primary cultured cardiomyocytes group were dividedinto 7groups, including normal group (Normal), aconitine incubated group(ACO), alkaline phosphatase treated group (A.P.), AAP treated group(AAP), aconitine incubated after AAP treated group (AAP+ACO), G(o|¨)6976 group (PKCαdepressant, G(o|¨) 6976) and AAP and G(o|¨) 6976 co-treatedgroup (AAP+G(o|¨) 6976). Extract total protein of cardiomyocytes ofexperimental groups and control group, and quantify the total protein byBCA standard protein regression curve. Quantify the change of expressionamounts of P-Cx43 (Ser-368), NP-Cx43 (Ser-368) and total PKCαandP-PKCα(Ser-657) in groups of cultured cardiomyocytes byWestern-bloting with anti-P-Cx43 (Ser-368) antibody, anti-NP-Cx43(Ser-368) antibody and anti-PKCαantibody and anti-P-PKCα(Ser-657)antibody. All the steps repeated 6 times.
3.5 Influence of the PKCαand P-PKCα(Ser-657) locusphosphorylation state in cardiomyocytes after aconitine incubation.
Anti-rabbit-anti-rat PKCαpolyclonal antibody (dilution at 1:200),rabbit anti-mouse phosphorylation PKCα(Ser-657) functional antibody(dilution at 1:200), Fluorescein mark goat via rabbit IgG and Rhodaminemark goat via rabbit IgG (dilution at 1:100) were selected to detect thespecific phosphorylation state of P-PKCα(Ser-657) locus with laserscanning confocal microscope and cellular image fluorescent locationanalysis technique before and after aconitine incubation.
3.6 Influence of the expression of calmodulin in aconitine culturedrats' cardiomyocytes.
Multiplex fluorescence reaction system and amplification methodwere set up with Na~+-Ca~(2+) crossover (NCX), phosphoric receptor sodiumalbumen (PLB), sarcoplasmic reticulum calcium pump Ca~(2+)-ATP ase(SERCA_2), Ryanodine receptor (RyR_2) and housekeeping gene (β-actin)by fluorescence labeling technique and RT-PCR in 12 wells of primarycultured cardiomyocytes groups. Capillary electrophoresis was used by3100 DNA Sequencer to detect the difference and change of transcriptmRNA of NCX, SERCA_2, RyR_2 and PLB in aconitine cultured groups andcontrol group. All the steps repeated 6 times.
3.7 Analyzing forensic postmortem examination documents of 10aconitine poisoning cases.
Collect forensic postmortem examination documents of 10 aconitinepoisoning cases, including systematic forensic anatomical data andhistopathological outcomes, investigation documents, rescue record and soon for some of the cases. Sort the data by age, gender, poisoning cause,poisoning type, and pathological change, characteristic and so on.Summarize the toxicological pathologic change of aconitine poisoning and investigate attention of forensic identification for aconitine poisoning.
3.8 Statistical analysis of experimental data.
Data from the study analyzed by SSPS statistical software (version10.0, USA) were expressed as (?)±s.
●Outcomes of comet assay were processed by CASP analysis software.Experimental data, such as head DNA (HDNA %), tail DNA(TDNA %), tail length (TL), tail moment (TM) and Olive tail moment(OTM) were measured and average value and standard deviation werecalculated and expressed by (?)±s. Each dosage group was comparedby one-way analysis of variance (ANOVA) with homogeneity ofvariance, and differences between groups were analyzed.
●The differences between expressions of P-Cx43 (Ser-368) andNP-Cx43 (Ser-368), relative expression amounts of total PKCαandP-PKCα(Ser-657) from control group and each experimental groupwere analyzed with univariate two-way (ANOVA) by SSPS statisticalsoftware; and multiple comparisons were made using Games-Howellpost hoc test.
●The difference and change of transcript mRNA of NCX, SERCA_2,RyR_2 and PLB in aconitine cultured groups and control group wereanalyzed by t-test with SPSS statistical software.
4 Results
4.1 Primary cardiomyocytes culture and condition optimization.
When plated in culture well, the cardiomyocytes were bright pellets inshape and suspended in medium. After 12 hours, they began to crawl onthe bottom of the culture well and spontaneous beating could be found infew cells. After 24 hours, attached myocytes connected each other, looked like a net; and slow synchronous beating could be found in all cultures.After 48 hours, the cardiomyocytes stretched on the bottom of culturewells, and high-speed (80～120 times per minute) synchronous beatingmonolayers of syncytium were formed. On 6th day of culture, meanlivability of cardiomyocytes were 97.33 % using trypan blue stainingmethod. Applying immunofluorescent microscopy with specificmonoclonal anti-cTnI antibody, we estimated that the mean purity ofcultured cardiomyocytes were 97.1%.
4.2 Setting-up models of cultured cardiomyocytes treated by aconitineand observing the toxic effect.
Aconitine incubated models were set up and it was verified by H.E.staining that the morphological structure of cardiomyocytes were hardlychanged after incubation with different dosages of aconitine compared tothe control cultures. But cardiomyocytes showed asynchronic beat,slowing beat and even arrest. There were certain correlation amongtoxicological pathologic change, toxic dose and poisoning period.
4.3 Observation of DNA damage of cultured rats' cardiomyocytesafter aconitine incubation.
After different concentrations of aconitine incubation, tail DNA, taillength, tail moment ~(TM) and Olive tail moment of cultured neonatal rats'cardiomyocytes increased, and head DNA decreased while theconcentration of aconitine increased. And there were all extremelysignificant differences compared with the control group (P＜0.01). Theoutcomes indicated that higher the dose of aconitine, severer the DNAdamage of cardiomyocytes.
4.4 The influence of expression of PKCαin cultured cardiomyocytes.
4.4.1 Observation of expression of P-Cx43 (Ser-368) in cardimyocytes.
Every experimental group was compared with the normal group. Andthe expressions of P-Cx43 (Ser-368) in cardiomyocytes with different treated patterns were not the same. The expression amounts of P-Cx43(Ser-368) in cardiomyocytes of ACO incubated group, A.R group and G(o|¨)6976 group decreased, and all increased in AAP group, AAP+ACO groupand AAP+G(o|¨) 6976 group. Games-Howell statistical method was selectedfor multiple comparisons of means between groups. Among ACOincubated group, A.P. group and G(o|¨) 6976 group, there was significantstatistical difference in ACO incubated group (P＜0.05), while there werehardly obvious difference among AAP group, AAP+ACO group andAAP+G(o|¨) 6976 group. The outcomes by Western Blotting presented thatafter ACO, A.R and G(o|¨) 6976 treated, the expression protein of Cx43(Ser-368) locus decreased, while after APP pre-treated increased obviously(P＜0.01).
4.4.2 Observation of expression of NP-Cx43(Ser-368) in cardiomyocytes.
The quantitative analytic outcomes of expression protein of NP-Cx43(Ser-368) locus phosphorylation state presented that the expression in A.P.group, ACO incubated group and G(o|¨) 6976 group reinforce (P＜0.01),especially A.P. group the most, while AAP group, AAP+ACO group andAAP+G(o|¨) 6976 group reduce (P＜0.01).
4.4.3 Observation of expression of total PKCαin cardiomyocytes.
The quantitative analytic result of total PKCαshowed that there wereno significant difference of expression of total PKCαin A.R group, ACOincubated group, G(o|¨) 6976 group, AAP group, AAP+ACO group andAAP+G(o|¨) 6976 group, compared with the control group (P＞0.05).
4.4.4 Observation of expression of total P- PKCα(Ser-657) incardiomyocytes.
Every experimental group was compared with the normal group. Andthe expressions of P-PKCα(Ser-657) in cardiomyocytes with differenttreated patterns were different. The P-PKCα(Ser-657) expressed incardiomyocytes of the normal group and ACO group, but none of A.Rgroup.
Compared with the normal group, the expressions of P-PKCα (Ser-657) phosphorylation protein in cardiomyocytes of ACO group andG(o|¨) 6976 group decreased obviously (P＜0.01). And the expressions ofphosphorylation increased in AAP group, AAP+ACO group and AAP+G(o|¨)6976 group (P＜0.01).
4.4.5 Observation of PKCαand PKCα(Ser-657) locus phosphorylationstate before and after aconitine incubation
Applying laser scanning confocal microscope, the immumofluorescenedetection results presented:
Total PKCαlabeled by Fluorescein, as green fluorescene signals, weredispersed distributed in cytoplasm of cardiomyocytes. There were nosignificant differences in ACO incubated group, G(o|¨) 6976 group, AAPgroup, AAP+ACO group and AAP+G(o|¨) 6976 co-treated group comparedwith the normal group.
Total P-PKCα(Ser-657) labeled by Rhodamine red fluorescene signalslabeling, as red fluorescene signals, were dispersed distributed incytoplasm of cardiomyocytes. The red fluorescene signals weakenedobviously and there were extremely significant differences in ACOincubated group and G(o|¨) 6976 group compared with the normal group(P＜0.01). There were also extremely significant differences of redfluorescene signals in AAP group, AAP+ACO group and AAP+G6 6976co-treated group (P＜0.01).
4.5 Influence of expression of calmodulin in aconitine cultured rats'cardiomyocytes.
It was detected that mRNA of RyR_2 and NCX expressed more, andPLB and SERCA_2 less by RT-PCR combined with multiplex fluorescence.
4.6 Analysis of forensic postmortem examination documents ofaconitine poisoning cases.
Characteristic of pathological changes were mainly that:①myocardium focal hemorrhage, transverse striation of cardiomyocytes wasunclear, sarcoplasm condensed and intercellular congestion;②focal or spotty necrosis of hepatocytes and fatty degeneration or hydropicdegeneration;③focal or globular hemorrhage of lung, focalpneumonedema;④scattered globular hemorrhage in gastric mucosa ofsome cases;⑤globular hemorrhage or scattered necrosis in kidney of afew cases.
5 Conclusions
●5.1 Optimization and specify cardiomyocytes culture method,establishment of aconitine incubated in vitro neonatal rats' cell modeland reference for forensic toxicologic study.
●5.2 DNA damage of aconitine incubated cardiomyocytes was ofobvious dose-effect relation, which would indicate that DNA damageplayed a role in toxic effect mechanism.
●5.3 Aconitine incubation would influence phosphorylation state ofPKCαand phosphorylation expression of PKCα(Ser657) decreased,which further induced that phosphorylated Cx43 of cardiomyocytesweakened. It inferred that the change of phosphorylation state ofPKCαand Cx43 was one of the paths of the aconitine poisoning.
●5.4 Aconitine influenced the expression of calmodulin incardiomyocytes, and mRNA of RyR_2 and NCX expressed more andPLB and SERCA_2 less. That calmodulin take part in toxic effectmechanism deserves further research.
●5.5 Announcements of forensic identification for aconitine poisoningwere raised after collection of forensic postmortem examinationdocuments of 10 aconitine poisoning cases and summarize thecharacters of toxicological pathologic change.
引文
1．张益鹄，黄光照，郭智贤，等．湖北部分地区8610例法医尸检资料的回顾性研究[J]．法医学杂志．1988，4(4)：1-6．
2．刘良，刘艳，黄光照．389例不同年份的中毒尸检资料分析[J]．刑事技术．1999，(6)：16-18．
3．张益鹄．中国特色的法医毒理学[J]．法医学杂志．2004，20(2)：101-104．
4．熊丽娟．中药毒性理论探讨[J]．云南中医学院学报，2007，30(3)：20-23．
5．罗贯中．三国演义[M]．北京：人民文学出版社，1997，617-623．
6．符华林．我国乌头属药用植物的研究概况[J]．中药材，2004，27(2)：149-152．
7. Van Landeghem A A, De Letter E A, Lambert W E, et al. Aconitine involvement in an unusual homicide case [J]. Int. J. Legal Med., 2006, 121(3):214-219.
8．张仲景著．《金匮要略》[M]．人民卫生出版社，2004年，21-25
9．蔡建成．毒剧中药的临床应用与体会[J]．中医药学报，2004，32(4)：44-45
10．张世忠，昊博威．乌头碱与钾离子通道激动剂合用对离体大鼠心脏的正性肌力作用研究[J]．中国药理学通报，2001，17(5)：570-572．
11．王华灵，韩培秀，徐世明．乌头碱对癌症疼痛的治疗效果[J]．中国中西医结合杂志，1994，14(4)：219-223．
12. Hikino H, Takata H, Fujiwara M, et al. Mechanism of inhibitory action of Mesaconitine in acute inflammations[J]. Eur J Pharmac, 1982, (82):65-71.
13．黄光照，汪德文．法医毒理学 第3版[M]．北京：人民卫生出版社，2004，218-222．
14. Baselt RC. Disposition of Toxic Drugs and Chemicals in Man, 7th ed [M]. Biomedical publications, Foster City, 2004, 357-363.
15. Camps FE, Gradwohl's Legal Medicine, 3rd ed [M]. John Wright & Sons, Bristol, 1976.
16．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
17. Elliott SP. A case of fatal poisoning with the aconite plant: quantitative analysis in biological fluid. Sci Justice, 2002, (42) :111-115.
18．何兴元．急性乌头碱中毒56例临床分析[J]．中华医学研究杂志，2006，6(12)：1381．
19. Ravi P., Lorraine Y., Barry C., et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion. J Forensic Sci,2008, 53:491-494.
20. Lin CC, Chan TYK, Deng JF. Clinical features and management of herb-induced aconitine poisoning [J]. Ann. Emerg. Meal., 2004, 43: 574-579.
21. Moritz F, Compagnon P,Kaliszczak IG, et al. Severe acute poisoning with homemade aconitum napellus capsules: toxicokinetic and clinical data [J]. J. Toxicol-Clin. Toxic., 2005, 43: 873-876.
22．柳大勇，郭鹏毅，樊栓．乌头碱中毒致死分析1例[J]．刑事技术2007．(6)：59．
23．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
24．龚冬悔，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
25. Amran M S, Homma N, Hashimoto K. Pharmacology of KB-R7943: a Na+-Ca~(2+) exchange inhibitor [J]. Cardiovasc. Drug Rev., 2003, 21(4):255-276.
26. Amran M S, Hashimoto K, Homma N. Effects of sodium-calcium exchange inhibitors, KB-R7943 and SEA0400, on aconitine-induced arrhythmias in guinea pigs in vivo, in vitro, and in computer simulation studies [J]. J. Pharmacol. Exp. Ther., 2004.310(1): 83-89.
27. Yan LIU, Shi-Wei ZHANG, Man LIANG, Qian LIU, Liang LIU. Effects of Aconitine on [Ca~(2+)] Oscillation in Cultured Myocytes of neonatal rats. Journal of Huazhong University of Science and Technology. 2008,28(5):499-503.
28. Harberd N, Artavanis-Tsakonas S, Berridge MJ, Bourne H, Cantley L, Downward J, et al. Cell Communication. In: Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, eds. Molecular Biology of the Cell. Garland Science, 4th ed. New York, NY: Taylor & Francis Group, 2002, 831-906.
29. Fonres P., Ratel S., Lecomte D.. Pathology of arrythmogenic right ventricular cardiopathy autopsy study of forensic cases[J].J Forensic Sci, 1998,43(4):773-783.
30. James T N. Normal and abnormal consequences of apoptosis in the human heart, form postnatal morphogenesis to parocymal arrbymias [J]. Circulation, 1994,90(1):557-573.
31．雷怀成，宋道江，易建华，等．大鼠乌头碱中毒心肌细胞凋亡的研究．中国工业医学杂志．2004，17(6)：373-374．
32. Zhang S-W, Liu Y, Huang G-Z, et al. Aconitine alters connexin43 phosphorylation status and [Ca~(2+)] oscillation patterns in cultured neonatal rat ventricular myocytes [J]. Toxicol. In Vitro, 2007,21 (8): 1476-1485.
33. Axelsen LN, Stahlhut M, Mohammed S, et al. Identification of ischemia-regulated phosphorylation sites in connexin43: a possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123)[J]. J. Mol. Cell. Cardiol., 2006,40:790-798.
34. Beardslee MA, Lerner DL, Tadros PN, et al. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia [J]. Circ. Res., 2000, 87:656-662.
35. Matsushita S, Kurihara H, Watanabe M, et al. Alterations of phosphorylation state of connexin43 during hypoxia and reoxygenation are associated with cardiac function [J]. J. Histochem. Cytochem., 2006, 54: 343-353.
36. Wang SY, Wang GK. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell. Signal., 2003, 15:151-159.
1．张益鹄．中国特色的法医毒理学．法医学杂志．2004 20(2)：101-104．
2．黄光照．《法医毒理学》．第三版，2004年．
3．http://www.med66.com/new/53a273aa2009/200924qiji11135.shtml 21世纪毒理学的发展趋势．2009年2月1日．
4．鄂征主编．《组织培养和分子细胞学技术》．北京出版社，第二版，1995年．
5. Wenzel KG, Wheatley JW, Don Byrd G, et al. Effect of nicotine on cultured rat heat cells [J]. Toxicol. Appl. Pharmacol., 1970, 17: 774-785.
6. Limaye DA, Shaikh ZA. Cytotoxicity of cadmium and characteristics of its transport in cardiomyocytes [J]. Toxicol. Appl. Parmacol., 1999, 154: 59-66.
7. Acosta D, Ramos K, Cardiotoxicity of tricyclic antidepressants in primary cultures of rat myocardial cells [J]. J. Toxicol. Environ. Health, 1984, 14: 137-143.
8. Estevez MD, Wolf A, Schramm U. Effect of PSC 833, Verapamil and amiodarone on adriamycin toxicity in cultured rat cardiomyocytes [J]. Toxicol. In Vitro, 2000, 14:17-23.
9. Manabu N, Stuart WR, James EK, et al. Ouabain increases sarcoplasmic reticulum calcium release in cardiac myocytes [J]. J. Pharmacol. Exp. Ther., 2004, 308: 1181-1190.
10．章诗伟．乌头碱对培养新生大鼠心室肌细胞Connexin43蛋白磷酸化状态及其胞内[Ca~(2+)]振荡模式的影响[D]．华中科技大学同济医学院博士研究生毕业论文，2006：19-40．
11. Harary I, Farley B. In vitro studies of isolated beating heart cells[J]. Science, 1960,131 : 1674-1675.
12. Simpson N.H., Milner, A.E. and Al-Rubeai, M.. Prevention of hybridoma cell death by bcl-2 during sub optimal culture conditions[J]. Biotechnol. Bioeng, 1997,54: 1-16.
13. Yamashita N, Nishida M, Hoshida S, et al. Induction of manganese superoxide dismutase in rat cardiac myocytes increases tolerance to hypoxia 24 hours after preconditioning [J]. J. Clin. Invest., 1994, 94: 2193-2199.
14．王宁，李应东．乳鼠心肌细胞原代培养方法的改进[J]．中国心血管病研究，2007，5(12)：920-922．
15．王涛，余志斌，谢满江，等．新生大鼠心肌细胞培养技巧[J]．第四军医大学学报，2003，24(2)：92．
16．陈淳媛，孙跃女，杨作威，等．新生大鼠心肌细胞原代培养[J]．实用预防医学，2007，14(4)：983-985．
17. Ray M, Srivastava S, Maitra S, et al. The hamster heart is resistant to calcium paradox [J]. Pharmacol. Res., 2000, 41:475-481.
18. Harary I, Farley B. In vitro studies on single beating rat heart cells [J]. Cell Res. 1963, 29: 451-474.
19. Blondel B, Roijem I, Cheneval JP.Heart cells in culture: a simple method for increasing the proportion of myoblasts [J]. Exprerientia 1971, 27: 356-358.
20. Grynberg A, Athias P, Degois M. Effect of change in growth environment on cultured myocardial cells investigated in a standardized medium [J]. In Vitro Cell. Dev. Biol. 1986, 22: 44-50.
21. Hoshida S, Nishida M, Yamashita N, et al. Heme oxygenese-1 expression and its relationto oxidative stress during primary culture of cardiomyocytes [J]. J. Mol. Cell Cardiol., 1996, 28: 1845-1855.
22. Saffitz JE, Green KG, Kraft WJ, et al. Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium [J]. Am. J. Physiol. Heart Circ. Physiol., 2000, 278:H1162-H1170.
23. Zeevi-Levin N, Barac YD, Reisner Y, et al. Gap junctional remodeling by hypoxia in cultured neonatal rat ventricular myocytes [J]. Cardiovasc. Res., 2005, 66: 64-73.
24．李润琴，慕晓玲．新生大鼠心肌细胞培养技术[J]．山西医药杂志，2007，36(2)：103-105．
25. Orita H, Fukasawa M, Hirooka S, et at. Cytoprotective effects of nicorandil on hypothermic injury to immature cardiac myocytes [J]. Jpn. Circ. J., 1994, 58: 653-661.
26. Mark GE, Strasser FF. Pacemaker activity and mitosis in cultures of newborn rat heart ventricle cells [J]. Exp. Cell Res., 1966, 44: 217-233.
27. Nagy JI, Li WEI, Roy C, et al. Selective monoclonal antibody recognition and cellular localization of an unphosphorylated form of connexin43 [J]. Exp. Cell Res., 1997, 236: 127-36.
28. Ross PD, McCarl RL. Oxidation of carbohydrates and palmitate by intact cultured neonatal rat heart cells [J]. Am. J. Physiol., 1984, 246: H389-H397.
29. Simpson P, Savion S, Differentiation of rat myocytes in single cell cultures with and without proliferating non-myocardial cells [J]. Circ. Res., 1982, 50:101-116.
30. Karl S, Christoph G, Thomas F, et al. Inhibition of the ubiquitin-proteasome pathway induces differential heat-shock protein response in cardiomyocytes and renders early cardiac protection [J]. Biochem. Bioph. Res. Co., 2002, 291: 542-549.
31. Stahlhut M, Petersen JS, Hennan JK, et al. The antiarrhythmic peptide rotigaptide (ZP132) increases connexin 43 protein expression in neonatal rat ventricular cardiomyocytes [J]. Cell. Commun. Adhes., 2006, 13:21-27.
32. L(u|¨)ss H, Schmitz W, Nermann J. A proteasome inhibitor confers cardioprotection [J]. Cardiovasc. Res., 2002, 54: 140-151.
33. Marengo FD, Wang S, Wang B, Langer GA. Dependence of cardiac cell Ca~(2+) permeability on sialic acid-containing sarcolemmal gangliosides [J]. J. Mol. Cell Cardiol., 1998, 30: 127-137.
34. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limitedby extra-cellular regulated kinases 1/2-mediated induction of cyclooxygenase-2 [J]. J. Biol. Chem., 1999, 274: 5038-5046.
35. Brooks G, Poolman RA, McGill CJ, et al. Expression and activities of cyclins and cyclin-dependent kinases in developing rat ventricular myocytes [J]. J. Mol. Cell Cardiol., 1997, 29: 2261-2271.
36. Wagner DR, Combes A, McTiernan Ch, et al. Adenosine inhibits lipopoly-saccharide induced cardiac expression of tumor necrosis factor-α[J]. Circ. Res., 1998, 82: 47-56.
37. Rubin Y, Kesslericekson G, Navon G. The effect of furosemide on calcium ion concentration in myocardial-cells [J]. Cell. Calcium, 1995, 18:135-139.
38. Matos MJ, Post JA, Roelofsen B, et al. Composition and organization of sarcolemmal fatty acids in cultured neonatal rat cardiomyocytes [J]. Cell. Biol. Int. Rep., 1990, 14: 343-352.
39. Tanka M, Ito H, Adachi S, et al. Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes [J]. Circ. Res., 1994, 75: 426-433.
40. Yasui K, Kada K, Hojo M, et al. Cell-to-cell interaction prevents cell death in cultured neonatal rat ventricular myocytes [J]. Cardiovasc. Res., 2000, 48: 68-76.
41．薛庆善．体外培养的原理与技术[M]．北京：科学出版社，2001，618-636．
42．杨晓宁，田宗文，黄焕斌，等．新生大鼠心肌细胞的体外培养[J]．解剖学杂志，2005，28(3)：361-362．
43．宋学立，钱令嘉．大鼠心肌细胞的培养纯化及其在药理毒理学中的应用[J]．实用医药杂志，2002，19(12)：950-951．
1．黄光照，汪德文主编．法医毒理学[M]．第3版．北京：人民卫生出版社，2004，218-222．
2．柳大勇，郭鹏毅，樊栓良．乌头碱中毒致死分析1例[J]．刑事技术，2007，(6)：59．
3. Elliott SP.A case of fatal poisoning with the aconite plant: quantitative analysis in biological fluid[J]. Sci Justice, 2002, 42:111-115.
4. Ravi P,Lorraine Y, Barry C, et al. A Case of fatal aconitine poisoning by monkshood ingestion[J]. J Forensic Sci,2008,53:491-494.
5．苏平，刘明俊．大鼠乌头碱中毒心肌超微结构的改变[J]．西安医科大学学报．1991，12(4)：321-324．
6．张桥主编．卫生毒理学基础[M]．第三版．北京：人民卫生出版社，2001：236-240．
7．刘世新，刘艳主编．实用生物组织学技术(21世纪高等医药院校教材)[M]．北京：科学出版社，2004年3月．50-146．
8．吴景录．32例急性乌头碱中毒的临床总结．中外健康文摘·医药卫生版，2007，4(7)：54-56．
9．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
10．蔡琪琳．急性乌头碱中毒10例临床分析[J]．中国中西医结合急救杂志，2007，14(1)：59．
11．张丽娟，高顺梅．乌头碱类中毒的临床观察及护理体会[J]．黑龙江医学，2002，26(2)：139．
12．赵庆桃．乌头碱中毒45例分析[J]．中国误诊学杂志，2004，4(1)：121-122．
13．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
14．杨庆君，尹琼，万书平．乌头碱中毒致心律失常10例临床分析fJ]．中国中西医结合急救杂志，2007，14(5)：274．
15．杨维明，寿天佑．乌头碱中毒致室性心律失常16例诊治分析[J]．浙江临床医学．2006，8(10)：1072．
16．林津，徐红，徐文仪，等．重度乌头碱中毒心律失常的抢救体会[J]．内科急危重症杂志．2002，8(4)：219-220．
17．邹华．一起乌头碱引起12人食物中毒的调查[J]．中国饮食卫生与健康．2005，3(5／6)：50-51．
18．施玉樑，魏仁榆，周念辉，等．乌头碱对神经肌肉接头电活动和神经干复合电位作用的观察[J]．生理学报，1980，32(2)：135-142．
19．张宏顺．乌头类中药毒性及中毒处理[J]．药物不良反应杂志，2005，7(2)114-115．
20．卢中秋，胡国新．乌头碱急性中毒及诊治研究现状[J]．中国中西医结合急救杂志，2005，12(2)：119-121．
21. Wang S Y, Wang G K. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell Signal, 2003,15(2): 151 - 159.
22．龚冬梅，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
23. Moric E, Herbert E, Trusz-Gluza M, et al. The implications of genetic mutations in the sodium channel gene (SCN5A) [J]. Europace, 2003, 5(4): 325-334.
24．刘岩．乌头类生物碱心肌毒性作用的研究[D]．沈阳：沈阳药科大学，2007，42-49．
25. Dhein S. Pharmacology of gap junctions in the cardiovascular system [J]. Cardiovasc Res, 2004, 62: 287-298.
26. Van Veen TAB, Van Rijen HV, Opthof T. Cardiac gap junction channels modulation of expression and channel properties [J]. Cardiovasc. Res., 2001, 51: 217-229.
27. Gutstein DE, Morley GE, Tamaddon H, et al. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43 Circ. Res., 2001, 88: 333-339.
28. Kl(?)ber AG. Mechanisms of ventricular arrhythmias: a perspective [J]. J. Cardiovasc. Pharmacol., 1991, 17:S1-S8.
29. Peters NS, Coromilas J, Severs NJ, et al. Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia [J]. Circulation, 1997, 95: 988-996.
30. Zhang S-W, Liu Y, Huang G-Z, et al. Aconitine alters connexin43 phosphorylation status and [Ca~(2+)] oscillation patterns in cultured neonatal rat ventricular myocytes [J]. Toxicol. In Vitro, 2007, 21(8): 1476-1485.
1. Fairbairn DW, Olive PL , O'Neill KL. The comet assay : a comprehensive review[J]. Mutat Res , 1995, 339 (1) :37 - 59.
2．王静，万树青．彗星试验在环境毒理与农药遗传毒性研究中的应用展望[J]．生物技术通报．2003，第1期：10-12．
3. Van Landeghem A A, De Letter E A, Lambert W E, et al. Aconitine involvement in an unusual homicide case [J]. Int. J. Legal Med., 2006, 121(3):214-219.
4. Ravi P., Lorraine Y., Barry C., et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion. J Forensic Sci, 2008; 53:491-494.
5. Lin CC, Chan TYK, Deng JF. Clinical features and management of herb-induced aconitine poisoning [J].Ann. Emerg. Med., 2004, 43: 574-579.
6．柳大勇，郭鹏毅，樊栓．乌头碱中毒致死分析1例[J]．刑事技术2007年，第6期：59．
7．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
8．黄光照，汪德文．法医毒理学[M]．第3版．北京：人民卫生出版社，2004：218-222．
9．龚冬梅，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
10. Amram M S, Hashimoto K, Homma N. Effcets of sodium-calcium exchang inhibitors, KB-R7943 and SEA0400, on acontine-induced arrhythmias in guinea pigs in vivo, in vitro, and in computer simulation studies[J]. J Pharmacol Exp Ther, 2004, 310: 83-89.
11．刘艳，朱传红，邓立斌，等．毒鼠强诱导细胞DNA损伤的彗星电泳检测[J]．中国法医学杂志，2004，19(5)：278-279．
12. Konca K, Lankoff A, Banasik Anna, et al. A cross-platform public domain PC image-analysis program for the comet assay[J]. Mutat Res, 2003, 534: 15-20.
13．张家放主编．医用多元统计方法[M]。湖北：华中科技大学出版社．2002：39-59．
14．高丰，金天明，姜宁，等．细胞凋亡的研究现状与发展趋势[J]．动物医学进展，2003，24(6)：1-3．
15. Wang JYJ. DNA damage and apoptosis[J]. Cell and Differentiation. 2001,8(11):1047-1048.
16. Chris J N , Boris Z. DNA damage-induced apoptosis[J]. Oncogene (2004) 23, 2797-2808.
17. Roos WP, Kaina B. DNA damage-induced cell death by apoptosis[J].Trends Mol Med. 2006, 12(9):440-450.
18. Gabriela F. R, Manuela CR, Bj(o|¨)rn J. Characterization of DNA Damage in Yeast Apoptosis Induced by Hydrogen Peroxide, Acetic Acid, and Hyperosmotic Shock. Molecular Biology of the Cell.2006, 17 (10): 4584-4591.
19．雷怀成，宋道江，易建华，等．大鼠乌头碱中毒心肌细胞凋亡的研究[J]．中国工业医学杂志．2004，17(6)：373-374．
20. Nakayama T, Kaneko M, Kodama M, et al. Volatile gas components contribute to cigarette smoke induced DNA single strand breaks in cultured humancells[J]. Agric Biol Chem 1986;50:3219-3220.
21. Hruszkewycz AM and Bergtold DS. The 8-hydroxyguanine content of isolated mitochondria increases with lipid peroxidation[J]. Mutat Res. 1990, 244, 123-128.
22．刘晓麒，曹恩华．脂质过氧化引起的DNA损伤研究进展[J]．生物化学与生物物理进展，1994，21(3)：218-222．
23．李雨民，杨凤桐．DNA损伤修复与细胞凋亡[J]．国外医学-放射医学核医学分册．1999，23(3)：112-115．
24. Lu YC, Huang CC, Huang C J, et al. Effects of Antrodia camphorata on viability, apoptosis, [Ca2+] i, and MAPKs phosphorylation in MG63 human osteosarcoma cells[J].Drug Development research. 2007, 68(2):71-78.
25. Wang X , Chen S, Ma G , et al. Involvement of proinflammatory factors, apoptosis , caspase-3 activation and Ca2+disturbance in microglia activation-mediated dopaminergic cell degeneration[J]. Mech. Ageing Dev. , 2005, 126: 1241-1254.
26．王华，张天一，杨扬，等．三氧化二砷诱导黑色素瘤B16细胞凋亡机制[J]．江苏医药，2006，32(12)：1128-1129．
27．许惠玉，陈志伟，牛建昭，等．赤芍总苷诱导K562细胞凋亡及对线粒体膜电位和Ca~(2+)的影响[J]．中国组织工程研究与临床康复，2008，12(16)：3123-3126．
28．付敏．乌头碱致心律失常的细胞分子机制的实验研究[D]．北京中医药大学博士研究生学位论文．2007：56-72．
29. Eigel BN, Gursahani H, Hadley RW. Na+/Ca2+ exchanger plays a key role in inducing apoptosis after hypoxia in cultured guinea pig ventricular myocytes[J]. Am J Physiol Heart Circ Physiol, 2004, 287:1466-1475.
30. Rodriguez Tarduchy G, Sahuquillo AG, Alarcon B et al.Apoptosis but not other activation events is inhibited by a mutation in the transmembrane domain of T cell receptor beta that impairs CD3zeta association[J].J Biol Chem, 1996, 271(48): 30417-30425.
31. Yan Liu, Shi-Wei Zhang, Man Liang, et al. Effects of Aconitine on [Ca2+] Oscillation in Cultured Myocytes of neonatal rats[J]. J Huazhong Univ Sci Technolog, 2008, 28(5): 499-503.
32．肖琳，王松，王凡．应用彗星实验检测细胞DNA损伤的原理与方法．四川解剖学杂志，2005，12(1)：39-42．
33. Lin SM, Yang MH. Arsenic, selenium, and zinc in patients with Blackfoot disease[J]. Biol Trace Elem Res. 1988, 15:213-221.
34．邢彩虹，李桂兰，纪之莹，等．单细胞凝胶电泳图像分析软件的比较．毒理学杂志．2005，19(2)：141-143．
35．张建平．单细胞凝胶电泳技术及其应用．国外医学遗传学分册，1997，20(5)：231-234．
36. Lee E, Oh E, Lee J , et al. Use of the tail moment of the lymphocytes to evaluate DNA damage in human biomoitoring studies[J]. Toxicol Sci, 2004, 81(1): 121 - 132.
37. Singh R, Sram RJ, Binkova B, et al. The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans[J]. Mutat Res, 2007, 620: 83- 92.
38. Cebulska WA, Binkova B, Sram RJ, et al. Repair competence assay in studies of the influence of environmental exposure to c-PAHs on individual susceptibility to induction of DNA damage[J]. Mutat Res, 2007, 620: 155- 164.
39. Ostling O, Johanson, KJ. Microelectrophoretic study of radiation-induced DNA damage in individual mammalian cells[J].Biochem Biophys Res Commun. 1984,123(2):291-298.
40. Singh R, Sram RJ, Binkova B, et al. The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans[J]. Mutat Res, 2007, 620: 83- 92.
1. Sohl G.,Willecke K. Gap junctions and the connexin protein family[J]. Cardiovasc.Res, 2004,62:228-232.
2. Dhein S, Polontchouk L, Salameh JA.Pharmacological modulation and differential regulation of the cardiac gap junction proteins connexin 43 and connexin 40[J]. Biology of the Cell ,2002, 94: 409-422.
3. Dupont E.Matsushita T.Kaba RA Altered connexin expression in human congestive heart failure[J]. J Mol Cell Cardiol. 2001,33:359-371.
4．李霞，张存泰，王琳，等．心力衰竭时CX43的改变及其作用[J]．中国心脏起搏与心电生理杂志，2005，19(5)：404-406．
5．苏德淳，常志文，范书英．缝隙连接蛋白43在缺血性心律失常中的作用[J]．首都医科大学学报，2006，27：235-238．
6. Schu lz R,Heusch G. Connexin 43 and ischem icprecond ition ing .Card iovasc Res, 2004,62, 62:335-344.
7. Giepmans BN. Gap junctions and connexin-interacting proteins .Cardiovasc Res, 2004,622, 62(2) :233-245.
8. Salameh A. Life cycle of connexins:regulation ofconnexin sythesis and degradation[J] .Adv Cardiol. 2006,42, 42:57-70.
9. Saez JC,Retamal MA,Basilio D,et al. Connexin-based gap junction hemichannels:gating mechanisms[J] .Biochim Biophys Acta. 2005,1711, 1711:215-224.7. Baselt RC. Disposition of Toxic Drugs and Chemicals in Man, 7th ed [M]. Biomedical publications, Foster City, 2004, 357-363.
10. Lin H,Ogawa K,Imanaga I,et al. Alterations ofcormexin 43 in the diabetic rat heart[J] .Adv Cardiol. 2006,42, 42:243-254.
11．王荣；张存泰；王琳．缝隙连接蛋白Cx43的磷酸化对缝隙连接通讯的调控[J]．中国心血管杂志，2008，
12．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
13．赵庆桃．乌头碱中毒45例分析[J]．中国误诊学杂志，2004，4(1)：121-122．
14．蔡琪琳．急性乌头碱中毒10例临床分析[J]．中国中西医结合急救杂志，2007，14 (1)：59．
15．张丽娟，高顺梅．乌头碱类中毒的临床观察及护理体会[J]．黑龙江医学，2002，26(2)：139．
16．章诗伟，任杰林，张黎，等．乌头碱对培养新生心室肌细胞Connexin 43蛋白磷酸化的影响[J]．中国法医学杂志，2008，23(2)：29-32．
17. Beardslee MA, Lerner DL, Tadros PN, et al. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia [J]. Circ. Res., 2000, 87:656-662.
18. Turner MS, Haywood GA, Andreka P, et al. Reversible connexin 43 dephosphorylation during hypoxia and reoxygenation is linked to cellular ATP levels [J]. Circ. Res., 2004, 95: 726-733.
19. M(u|¨)ller A, Gottwald M, Tudyka T, et al. Increase in gap junction conductance by an antiarrhythmic peptide [J]. Eur. J. Pharmacol., 1997, 327:65-72
20. M(u|¨)ller A, Schaefer T, Linde W, et al. Actions of the antiarrhythmic peptide AAP10 on cellular coupling [J]. Naunyn. Schmiedebergs. Arch. Pharmacol., 1997, 356: 76-82.
21. Dhein S, Weng S, Polontehouk L, et al. Pharmacological modification of gap junctional coupling by antiarrhythmic peptides. Role of PKC [J]. Naunyn. Schmiedebergs. Arch. Pharmacol., 2001, 363:R99 [Suppl.].
22. Weng S, Lauven M, Schaefer T, et al. Pharmacological modulation of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation [J]. FASEB J. 2002, 16: 1114-1116.
23. Axelsen LN, Stahlhut M, Mohammed S, et al. Identification of ischemia-regulated phosphorylation sites in connexin43: a possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123) [J]. J. Mol. Cell. Cardiol., 2006, 40: 790-798.
24. Stahlhut M, Petersen JS, Hennan JK, et al. The antiarrhythmic peptide rotigaptide (ZP132) increases connexin 43 protein expression in neonatal rat ventricular cardiomyocytes [J]. Cell. Commun. Adhes., 2006, 13:21-27.
25. Matsushita S, Kurihara H, Watanabe M, et al. Alterations of phosphorylation state of connexin43 during hypoxia and reoxygenation are associated with cardiac function [J]. J. Histochem. Cytochem., 2006, 54: 343-353.
26. Dhein S, Weng S, Polontehouk L, et al. Pharmacological modification of gap junctional coupling by antiarrhythmic peptides. Role of PKC [J]. Naunyn. Schmiedebergs. Arch. Pharmacol., 2001, 363:R99 [Suppl.].
27. Weng S, Lauven M, Schaefer T, et al. Pharmacological modulation of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation [J]. FASEB J. 2002, 16: 1114-1116.
28. Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication [J]. Int. J. Biochem. Cell Biol., 2004, 36:1171-1186.
29．李喜涛，许文灿．连接蛋白43磷酸化状态与缝隙连接通道功能的关系．汕头大学医学院学报，2008，21(3)：185-188．
30．庄燕燕，唐雪莲．间隙连接及其磷酸化调节．国际病理科学与临床杂志．2006，26(5)：444-447．
31. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses [J]. Am. J. Physiol.-Lung C. 2000, 279: L429-L438.
32. Joell L S, Paul D L. Connexin phosphorylation as a regur latory event linked to gap junction channel assembly[J]. Biochimica et Biophysica Acta, 2005,1711(2):154-163.
33. EK-Vttop J F, King T J, Heyman N S, et al. Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation[J]. Circ Res, 2006.98:1498-1505.
34. Zeevi-Levin N, Barac YD, Reisner Y, et al. Gap junctional remodeling by hypoxia in cultured neonatal rat ventricular myocytes [J]. Cardiovasc. Res., 2005, 66: 64-73.
35. Saffitz JE, Green KG, Kraft WJ, et al. Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium [J]. Am. J. Physiol. Heart Circ. Physiol., 2000, 278:H1162-H1170.
36．张家放主编．医用多元统计方法[M]。湖北：华中科技大学出版社．2002：39-59．
37. Lampe PD, Lan AF.The effects of connexin phosphorylation on gap junctional communication[J]. Biochem Cell Biol,2004,36:1171-1186.
38. Beardslee MA, Lerner DL, Tadros PN, et al. Dephosphorylation and intracellular redistribution of ventricular connexin 43 during electrical uncoupoing induced by ischemial[J].Circ Res, 2000,87:656-662.
39. Guerrero PA, Schuessler RB, Davis LM, et al. Slow ventricular conduction in mice heterozygous for a connexin 43 null mutation[J]. J Clin Invest, 1997,99:1991-1998.
40. Thomas SA, Schuessler RB, Berul CI, et al. Disparate effects of deficient expression of connexin 43 on atrial and ventricular conduction : evidence for chamber-specific molecular determinations of conduction [J]. circulation, 1998, 97:686-691.
41. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses. Am. J. Physiol.-Lung C, 2000, 279:L429-L438.
42. Parekh DB, Ziegler W, Parker PJ. Muliple pathways control protein kinase C phosphorylation. EMBO J., 2000,19: 496-503.
43. Bornancin F, Parker PJ. Phosphorylation of protein kinase C-α on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state. J. Biol. Chem., 1997, 272: 3544-3549.
44. Weng S, Lauven M, Schaefer T, et al. Pharmacological modulation of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation [J]. FASEB J. 2002,16: 1114-1116.
45. Dhein S, Weng S, Grover R, et al. Protein kinase C alpha mediates the effect of antiarrhythmic peptide on gap junction conductance [J]. Cell Commun. Adhes., 2001,8:257-264.
46. Van Veen TAB, Van Rijen HV, Opthof T. Cardiac gap junction channels modulation of expression and channel properties [J]. Cardiovasc. Res., 2001, 51: 217-229.
47. Kumar NM, Gilula NB. The gap junction communication channel [J]. Cell, 1996, 84: 381-388.
48. Dhein S. Pharmacology of gap junctions in the cardiovascular system [J]. Cardiovasc Res, 2004, 62: 287-298.
49. Axelsen LN, Stahlhut M, Mohammed S, et al. Identification of ischemia-regulated phosphorylation sites in connexin43: a possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123) [J]. J. Mol. Cell. Cardiol., 2006, 40: 790-798.
50. Matsushita S, Kurihara H, Watanabe M, et al. Alterations of phosphorylation state of connexin43 during hypoxia and reoxygenation are associated with cardiac function [J]. J. Histochem. Cytochem., 2006, 54: 343-353.
51. Muller A, Gottwald M, Tudyka T, et al. Increase in gap junction conductance by an antiarrhythmic peptide [J]. Eur. J. Pharmacol., 1997, 327: 65-72.
52. Muller A, Schaefer T, Linde W, et al. Actions of the antiarrhythmic peptide AAP10 on cellular coupling [J]. Naunyn. Schmiedebergs. Arch. Pharmacol., 1997,356: 76-82.
53．王荣，张存泰，阮燕菲，等大鼠心肌急性缺氧时缝隙连接蛋白43磷酸化水平的改变及心律失常肽10对其影响[J]．临床心血管疾病杂志．2007，23(5)：375-377．
54. TenBroek EM, Lampe PD, Solan JL, et al. Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP[J]. J Cell Biol,2001,155:1307-1318.
55. Lampe PD. Analyzing phorbol ester effects on gap junction communication : A dramatic inhibition of assembly[J]. J Cell Biol, 1994,127:1895-1905.
56. Lin H, Ogawa K, Imanaga I, et al. Alteration of connexin43 in the diabetic rat heart[J]. Adc Cardiol, 2006,42:243-254.
57. Wagner LM, Saleh SM, Boyle DJ, et al. Effects of protein kinase Cgammma on gap junction disassembly in lens epithelial cellsand retinal cells in culture[J]. Mol Vis,2002,8:59-66.
58. Cruciani V, Sanner T, Mikalsen SO. Pharmacological evidence for system-depenmant involvement of protein kinase C isoenzymes in phorbol ester-suppressed gap junctional communication[J]. Carcino genesis, 200122:221-231.
59. Wang S Y, Wang G K. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell. Signal., 2003, 15(2): 151-159.
60．龚冬梅，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
61. Moric E, Herbert E, Trusz-Gluza M, et al. The implications of genetic mutations in the sodium channel gene (SCN5A) [J]. Europace, 2003, 5(4): 325-334.
62．刘岩．乌头类生物碱心肌毒性作用的研究[D]；沈阳药科大学；2007年，35-48．
63．苏平，刘明俊．大鼠乌头碱中毒心肌超微结构的改变[J]．西安医科大学学报．1991，12(4)：321-324．
64．李宏，刘明俊，陈念祖．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅰ)-线粒体细胞色素C氧化酶[J]．中国法医学杂志，1988，3(4)：199-201．
65．李宏，陈念祖，刘明俊．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅱ)-线粒体琥珀酸脱氢酶(SDH)的定位[J]．中国法医学杂志，1988，3(4)：202-205．
66．梁强荣，刘明俊，胡炳蔚．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅲ)-线粒体NADHD的组织化学和细胞化学研究[J]．中国法医学杂志，1991，6(2)：84-86．
67．梁强荣，刘明俊，胡炳蔚．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅳ)-乳酸脱氢酶的组织化学和细胞化学定位[J]．中国法医学杂志，1991，6(3)：140-141．
68. Yan LIU, Shi-Wei ZHANG, Man LIANG; Qian LIU, Liang LIU. Effects of Aconitine on Ca~(2+)] Oscillation in Cultured Myocytes of neonatal rats. Journal of Huazhong University of Science and Technology. 2008,28(5):499-503.
1. Berridge MJ, Bootman MD, Roderick HL. Calcium signaling:dynamics, homeostains and remodeling [J]. MOL Cell Biol. 2003, 4:517-529.
2. Anderson ME. Calmodulin and the philosopher's stone: changing Ca~(2+) into the arrhythmias[J].J Cardiovase Electrophysiol. 2002, 13:195-197.
3. NiuH L, Pan ZW, Zhu JX, eta.l Dihydromyricetin exerted its va-soconstrictive effects by increasing intracellular Ca~(2+) in isolated canine carotid artery[J].Chin Pharmacol Bull, 2007, 23 (11) :1437-40.
4．张高小，王玲，李超，等．细胞内钙在脑心综合征心律失常中作用研究．中国药理学通报．2008，24(12)：1607-1610．
5．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
6. Elliott SP. A case of fatal poisoning with the aconite plant: quantitative analysis in biological fluid. Sci Justice, 2002;42:111-115.
7．何兴元．急性乌头碱中毒56例临床分析[J]．中华医学研究杂志，2006，6(12)：1381．
8. Ravi P., Lorraine Y., Barry C., et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion. J Forensic Sci, 2008; 53:491-494.
9. Yan Liu, Shi-Wei Zhang, Man Liang, et al. Effects of Aconitine on [Ca~(2+)] Oscillation in Cultured Myocytes of neonatal rats[J]. J Huazhong Univ Sci Technolog, 2008, 28 (5) :499-503.
10．柯俊，张存泰，马业新，等．钙调蛋白激酶Ⅱ抑制剂对心肌肥厚兔室性心律失常的影响．中华心血管病杂志．2007，35(1)：33-35．
11. Marx SO, Gaburjakova J, Gaburjakova M, Henrikson C, Ondrias K, Marks AR. Coupled gating between cardiac calcium release channels(ryanodine receptors) [J]. Circ Res 2001, 88: 1151-1158.
12. Marx SO, Ondrias K, Marks AR. Coupled gating between individual skeletal muscle calcium release channels ( ryanodine receptors ). Science 1998, 281:818-821
13. Wu X D, Dai D Z, Zhang QP, et al. Proparnolol and vera-pamil inhibit Mrna expression of RyR2 and SERCA in L-thyroxin-induced rat ventricular hypertrophy[J].Acta Pharmacol Sin. 2004, 25 (3) : 347-351.
14. Wehrens XH, Marks AR. Altered function and regulation of cardiac ryanodine receptors in cardiac disease[J].Trends Biochem Sci, 2003, 28 (12) : 671-678.
15. Wehrens X H, Lehnart S E, Huang F, et al. FKBP12·6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death[J].Cell, 2003, 113 (7) : 829-840.
16．朱传红．毒鼠强中毒的法医分子毒理学研究[D]．华中科技大学同济医学院博士研究生学位论文，2005：91-106．
17．付敏．乌头碱致心律失常的细胞分子机制的实验研究[D]．北京中医药大学博士研究生学位论文，2007：97-120．
18．刘晓宇．Wnt对Connexin43表达及其功能的影响[D]．复旦大学博士研究生学位论文，2007：38-55
19. Glover G, Mitchell J. An Introduction To Biostatistics[M]. McGraw-Hill Companies, Inc. 2001: 296.
20．张家放主编．医用多元统计方法[M]。湖北：华中科技大学出版社．2002：39-59
21．崔婕，薛绍白．胞内钙稳态调节[J]．细胞生物学杂志，1995；17(3)；97-102．
22．祝宝华，奚群英，刘晨，等．细胞膜钠钙交换蛋白在大鼠心肌缺血预适应及腺苷诱导预适应中的作用及其信号转导[J]．中华心血管病杂志．2006，34(4)：
23. Hilgemann D W, Nicoll D A, Philipson K D. Change movement during Na~+ translocation by native and cloned cardiac Na~+/Ca~(2+) exchanger [J]. Nature, 1991, 352 (6337) :715-718.
24. Bridge J H B, Smolley J R, Spitzer K W. The relationship between charge movements associated with ICa and INa-Ca in cardiac myocytes [J]. Science, 1990, 248 (4953) :376-378.
25. Xander H, Andrew R. Altered function and regulation ofcardic ryanodine receptor in cardic disease[J]. Trends Bio Sci 2003, 28 (12) : 671-67.
26. Franzini-Armstrong C, ProtastF, RameshV. Shape size and distribution of Ca~(2+) release units and couplons in skeletal and cardiacmuscles[J]. Biophys J, 1999, 77: 1528-1539
27. MeissnerG. Ryanodine receptor /Ca~(2+) release channels and their regulation by endogenous effectors[J]. AnnuRev Physiol 1994, 56: 485.
28. WilliamsA, WestD, Sitsapesan R. Light at the end of thecalcium release channel tunne:1 Structures and mechanisms involved in ion translocation in ryanodine receptor channels[J]. Q Rev Biophys, 2001, 34: 61.
29. Xander H, Andrew R. Altered function and regulation of cardic ryanodine receptor in cardic disease[ J]. Trends Bio Sci , 2003, 28 (12) : 671-67.
30. Clark M, Leo T W, Maelcrman DH.Functional consequences of alterations to polar amino acids located in the mmsmembrane domain of the Ca2 (+) -ATPase of sareoplasmic reticulum[J]J Bioehem, 1990; 265 (11) : 6262-6267.
31．蒋艳伟，刘尔亮，朱少华，等．兰尼碱受体及其法医学意义[J]．中国法医学杂志．2008，23(1)：38-40．
32．李江，胡申江，赵晓燕，等．腺相关病毒载体介导的PLB基因反义RNA对大鼠心肌细胞肌浆网Ca~(2+)-ATPase活性和[Ca~(2+)]i的作用[J]．中国病理生理杂志．2006，22(1)：39-42．
33．孙祝美．心肌细胞与钙离子调控[J]．广州医药．2008，39(3)：1-3．
34．郑霞，孙坚，胡申江，等．缺血／再灌注心肌肌浆网肌钙调控蛋白mRNA表达的变化[J]．中国应用生理学杂志．2006，22(2)：142-146．
35. Matolweni L O, Bardien S, Rebello G, et al. Arrhythmogenic right ventricular cardiomyopathy type 6 (ARVC6) : sup-port for the locus assignment , narrowing of the criticalregion and mutations creening of three candidate genes [J].BMC Med Genet, 2006, 28 (7) :29.
36. Akao Y, Nakagawa Y, AkiyamaK. Arsenic trioxide induces apoptosis in neuroblastoma cell lines through the activation of caspase-3 in vitro [J]. FEBS Lett, 1999, 455 (1-2) :59-62.
37. Yung HC, Man KH, Chong HR, et al. Induction of apoptotic cell death by mycelium extracts of Phellinus linteus in human neuroblastoma cells[J]. International Journal Of Molecular Medicine. 2004, 14: 227-232.
38. Ananth C., Thameem-Dheen S, Gopalakrishnakone P, et al. Domoic acid-induced neuronal damage in the rat hippocampus: Changes in apoptosis related genes (Bcl-2, Bax, caspase-3 ) and microglial response[J]. Journal of Neuroscience Research.2001, 66 (2) :177-190.
39. He CH, Waxman AB, Lee CG, et al. Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury[J]. J Clin Invest. 2005, 115:1039-1048.
40. Jayanthi S, Deng XL, Noailles P H, et al. Methamphetamine induces neuronal apoptosis via cross-talks between endoplasmic reticulum and mitochondria-dependent death cascades[J]. The FASEB Journal. 2004, 18:238-251.
41. Ananth, Dheen ST, Gopalakrishnakone P, et al. Distribution of NADPH-diaphorase and expression of nNOS, N-methyl-D-aspartate receptor (NMDAR1) and non-NMDA glutamate receptor (GlutR2) genes in the neurons of the hippocampus after domoic acid-induced lesions in adult rats[J]. Hippocampus. 2003, 13 (2): 260-272.
42．郭景元，李伯龄主编．中国刑事技术大全法医物证学分册[M]．北京：中国人民公安大学出版社出版．2002，335-355．
1．张益鹄．中国特色的法医毒理学[J]．法医学杂志，2004，20(2)：101-104．
2．熊丽娟．中药毒性理论探讨[J]．云南中医学院学报，2007，30(3)：20-23．
3．顾清光．神农本草经[M]．学苑出版社，2007：27．
4．张仲景．金匮要略[M]．人民卫生出版社，2004：21-25．
5．蔡建成．剧毒中药的临床应用与体会[J]．中医药学报，2004，32(4)：44-45．
6．云南省卫生厅．关于食用添加乌头碱类药物中毒的预警公告[EB／OL]．http://www. yn.chinanews.com.cn/news/html/tingjudongtai/shengweishengting/20081117/76000.html, 2008-11-17.
7．付静．食用草乌谨防乌头碱中毒[N]．保健时报，2009-2-5(002)．
8．黄光照，汪德文．法医毒理学(第3版)[M]．北京：人民卫生出版社，2004：218-222．
9．苏建宏，余成敏．68例乌头碱中毒的心电图分析[J]．中国医疗前沿，2009，4(3)：104．
10. Baselt RC. Disposition of Toxic Drugs and Chemicals in Man, 7~(th) Ed [M].Biomedical publications, Foster City, 2004, 357-363.
11. Camps FE. Gradwohl's Legal Medicine, 3~(rd) Ed [M]. John Wright & Sons, Bristol, 1976, 215-236.
12．陈安宝，陈嘉勇，梁道明，等．急性中毒2658例流行病学分析[J]．中国急救医学，2008，28(5)：409-411．
13. Van Landeghem AA, De Letter EA, Lambert WE, et al. Aconitine involvement in an unusual homicide case [J]. Int. J. Legal Med., 2006, 121(3): 214-219.
14. Ravi P., Lorraine Y., Barry C., et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion [J]. J Forensic Sci, 2008, 53: 491-494.
15. Lin CC, Chan TYK, Deng JF. Clinical features and management of herb-induced aconitine poisoning [J]. Ann. Emerg. Med., 2004, 43: 574-579.
16．柳大勇，郭鹏毅，樊栓．乌头碱中毒致死分析1例[J]．刑事技术，2007，6：59．
17．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
1．黄光照，汪德文．法医毒理学[M]．第3版．北京：人民卫生出版社，2004，218-222．
2. Elliott SP.A case of fatal poisoning with the aconite plant: quantitative analysis in biological fluid [J]. Sci Justice, 2002, 42:111-115.
3. Pullela R, Young L, Gallagher B, et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion [J]. J Forensic Sci, 2008, 53: 491-494.
4．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
5. Mori A, Mukaida M, Ishiyama I, et al. Homicidal poisoning by aconite: report of a case from the viewpoint of clinical forensic medicine [J]. Nihon Hoigaku Zasshi, 1990, 44(4): 352-357.
6．张益鹄．中国特色的法医毒理学[J]．法医杂志学，2004，20(2)：101-104．
7．符华林．我国乌头属药用植物的研究概况[J]．中药材，2004，27(2)：149-152．
8. Van Landeghem AA, De Letter EA, Lambert WE, et al. Aconitine involvement in an unusual homicide case [J]. Int J Legal Med., 2006, 121(3): 214-219.
9．熊丽娟．中药毒性理论探讨[J]．云南中医学院学报，2007，30(3)：20-23．
10．张仲景．金匮要略[M]．北京：人民卫生出版社，2004，
11．蔡建成．毒剧中药的临床应用与体会[J]．中医药学报，2004，32(4)：44-45．
12．王华灵，韩培秀，徐世明．乌头碱对癌症疼痛的治疗效果[J]．中国中西医结合杂志，1994，14(4)：219-223．
13．张世忠，吴博威．乌头碱与钾离子通道激动剂合用对离体大鼠心脏的正性肌力作用研究[J]．中国药理学通报，2001，17(5)：570-572．
14. Hikino H, Takata H, Fujiwara M, et al. Mechanism of inhibitory action of Mesaconitine in acute inflammations [J]. Eur J Pharmacol, 1982, 82(1-2): 65-71.
15．柳大勇，郭鹏毅，樊栓．乌头碱中毒致死分析1例[J]．刑事技术，2007，31(6)：59．
16．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
17．吕桂玲，张一民，宋兴芳，等．653例含乌头碱类药物致不良反应文献分析[J]．中国药房，2007，18(5)：374-376．
18．赵庆桃．乌头碱中毒45例分析[J]．中国误诊学杂志，2004，4(1)：121-122．
19．阮海林，陆宁．乌头碱中毒的特点及治疗(附25例临床分析)[J]．广西医科大学学报，2000，17(1)：160．
20．朱永福，宋江萍，周永丽．乌头碱类中药中毒致心律失常22例[J]．中国危重病急救医学，2000，12(3)：136．
21. Yeih DF, Chiang FT, Huang SK, et al. Successful treatment of aconitine induced life threatening ventricular tachyarrhythmia with amiodarone [J]. Heart, 2000, 84(4): E8.
22．张丽娟，高顺梅．乌头碱类中毒的临床观察及护理体会[J]．黑龙江医学，2002，26(2)：139．
23．吴景录．32例急性乌头碱中毒的临床总结．急性乌头碱中毒的临床总结[J]．中外健康文摘(医药月刊)，2007，4(7)：54-56．
24．张丽，李晔．乌头碱中毒致精神障碍2例[J]．包头医学院学报，2002，18(3)：245．
25. Ohuchi S, Izumoto H, Kamata J, et al. A case of aconitine poisoning saved with cardiopulmonary bypass [J]. Kyobu-Geka, 2000, 53(7): 541.
26．徐红，林津．重度乌头碱中毒致血红蛋白尿2例[J]．内科急危重症杂志，2002，8(3)：12．
27．赵初环，卢中秋，黄唯佳，等．血液净化治疗急性乌头碱中毒[J]．中华内科杂志，2001，40(7)：50．
28．苏平，刘明俊．大鼠乌头碱中毒心肌超微结构的改变[J]．西安医科大学学报，1991，12(4)：321-324．
29. Skrupskii V A, Plaksin S E. Changes in the fatty acid composition of the phospholipids in the internal organs of rats during the modeling of aconitine arrhythmia [J]. Eksp. Klin. Farmakol., 1994, 57(4): 53-55.
30．李宏，刘明俊，陈念祖．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅰ)-线粒体细胞色素C氧化酶[J]．中国法医学杂志，1988，3(4)：199-201．
31．李宏，陈念祖，刘明俊．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅱ)-线粒体琥珀酸脱氢酶(SDH)的定位[J]．中国法医学杂志，1988，3(4)：202-205．
32．梁强荣，刘明俊，胡炳蔚．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅲ)-线粒体NADHD的组织化学和细胞化学研究[J]．中国法医学杂志，1991，6(2)：84-86．
33．梁强荣，刘明俊，胡炳蔚．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅳ)-乳酸脱氢酶的组织化学和细胞化学定位[J]．中国法医学杂志，1991，6(3)：140-141．
34．陈啸梅．组织化学手册[M]．第1版．北京：人民卫生出版社．1982，220-222．
35．陈忆九，乌仁，黄晓华．心脏性猝死心肌细胞凋亡研究[J]．法医学杂志，1999，15(3)：133-134．
36. Fonres P, Ratel S, Lecomte D. Pathology of arrythmogenicri ghtve ntricular cardiopathy autopsy study of forensic cases [J]. J Forensic Sci, 1998, 43(4): 773-783.
37. James TN. Normal and abnormal consequences of apoptosis in the human heart. Form postnatal morphogenesis to paroxymal arrhythmias [J]. Circulation, 1994, 90(1): 557-573.
38．雷怀成，宋道江，易建华，等．大鼠乌头碱中毒心肌细胞凋亡的研究[J]．中国工业医学杂志，2004，17(6)：373-374．
39. Wang SY, Wang GK. Voltage-gated odium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell Signal, 2003, 15(2): 151-159.
40．龚冬梅，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
41. Moric E, Herbert E, Trusz-Gluza M, et al. The implications of genetic mutations in the sodium channel gene (SCN5A) [J]. Europace, 2003, 5(4): 325-334.
42. Gellens ME, George AL Jr, ChenLQ, et al. Primary strueture and funetional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel [J]. Proc Natl Acad Sci U S A, 1992, 89(2): 554-558.
43. George AL, Varkony TA, et al. Assignment of the human heart tetrodotoxin resistant voltage-dependent Na channel α-subunit gene (SCN5A) to band 3p21 [J]. Cytogenet Cell Genet, 1995, 68(1-2): 67-70.
44. Wang Q, Shen JX, Splawki I, et al. SCN5A mutations associated with an inherited cardiac arrhythmia, Long QT syndrome [J]. Cell, 1995, 80(5): 805-911.
45．刘岩．乌头类生物碱心肌毒性作用的研究[D]．沈阳药科大学，2007，35-48．
46. Benitah JP, Gomez AM, Faueonnier J, et al. Voltage-gated Ca~(2+) currents in the human pathophysiologic heart: a review [J]. Basic Res Cardiol, 2002, 97 Suppl 1: 111-18.
47．于雷．心肌离子通道的研究进展[J]．新血管病学进展，2006，27(4)：413-416．
48. Patterson E, Lazzara R, Szabo B, et al. Sodium-Calcium Exchange Initiated by the Ca2+ Transient: An Arrhythmia Trigger within Pulmonary Veins [J]. J Am Coll Cardiol, 2006, 47(6): 1196-1206.
49．陈龙，马骋，蔡宝昌，等．乌头碱对大鼠心肌细胞钙通道阻滞作用的单通道分析[J]．药学学报，1995，30(3)：168-171．
50．吴晓冬，戴德哉．雷诺定受体与心律失常[J]．东南大学学报(医学版)，2005，24(1)：55．
51. Wehrens XH, Lehnart SE, Huang F, et al. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death [J]. Cell, 2003, 113(7): 829-840.
52. Priori SG, Napolitano C, Tiso N, et al. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia [J]. Circulation, 2000, 103(2): 196-200.
53. Tiso N, Stephan DA, Nava A, et al. Identification of mutations in the cardiac ryanodine receptor gene in families afected with arrhythmogenic right ventricular cardiomyopathy type 2(ARVD2) [J]. Hum Mol Genet, 2001, 10(3): 189-194.
54. Laitinen PJ, Brown KM, Piippo K, et al. Mutations of the cardiac ryanodine receptor gene in familial polymorphic ventricular tachycardia [J]. Circulation, 2001, 103(4): 485-490.
55. Sipido KR, Volders PG, de Groot SH, et al. Enhanced Ca (2+)release and Na/Ca exchange activity in hypertrophied canine ventricular myocytes: potential link between contractile adaptation and arrhythmogenesis [J]. Circulation, 2000, 102(17): 2137-2144.
56. Wehrens XH, Lehnart SE, Reiken SR, et al. Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2 [J]. Science, 2004, 304(5668): 292-296.
57．杨琳，黄诒焯．心肌细胞离子通道与心律失常[J]．中国心脏起搏与心电生理杂志，2003，17(2)：85．
58．付敏．乌头碱致心律失常的细胞分子机制的实验研究[D]．北京中医药大学，2007．
59．郭佳．钾离子通道结构的新发现[J]．河南医学研究，2006，15(2)：189-192．
60. Brown AM. Drugs, hERG and sudden death [J]. Cell Calcium, 2004, 35(6):543-547.
61．夏敏，陈义汉．心脏钾离子通道相互作用的研究进展[J]．东南大学学报(医学版)，2004，23(4)：278-281．
62．刘水平，陈玉川．心脏钾离子通道的电生理和分子多样性[J]．湖北化民族学院学报：医学版，2001，18(2)：38-40．
63. Bosch RF, Zeng X, Grammer JB, et al. Ionic mechanisms of electrical remodeling in human atrial fibrillation [J]. Cardiovasc Res, 1999, 44(1): 121-131.
64. Karle CA, Zitron E, Zhang W, et al. Human carfliac inwardly-rectifying K~+ channel kir(2.1b) is inhibited by direct protein kinase dependent regulation in human isolated cardiomyocytes and in an expression system [J]. Circulation, 2002, 106 (12): 1493-1499.
65. Wickman KD, Iniguwz-Lluhi JA, Davenport PA, et al. Reconr binant G-protein β_γ-subunits activate the muscarinic- gated atrial potassium channel [J]. Nature, 1994, 368(6468): 255-257.
66. Liu Y, Xu CQ, Jiao JD, et al. M_3-R/I_K(M3)--a new target of antiarrhythmic agents [J]. Yao Xue Xue Bao, 2005, 40(1): 8-12.
67. Grover GJ, Garlid KD. ATP-Sensitive potassium channels: a review of their cardioprotective pharmacology [J]. J Mol Cell Cardiol, 2000, 32(4): 677-695.
68. Kaab S, Zwermann L, Barth A, et al. Selective block of sarcolemmal IKATP in human cardiomyocytes using HMR 1098 [J]. Cardiovasc Drugs Ther, 2003, 17 (5-6): 435-441.
69. Wang S.Y., Wang G.K.. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell signal, 2003, 15(2): 151 -159.
70. A.D. Zechnich, J.R. Hedges, D Eiselt-Proteau, et al. Possible interactions with terfenadine or astemizole [J]. West J Med, 1994, 160(4): 321-325.
71．董晞，赵世萍，刘岩，等．甘草苷对乌头碱致心肌细胞损伤的保护作用[J]．中华中医药杂志，2009，24(2)：163-166．
72. Hilgemann D W, Nicoll D A, Philipson K D. Change movement during Na~+ translocation by native and cloned cardiac Na~+/Ca~(2+) exchanger [J]. Nature, 1991, 352(6337): 715-718.
73. Bridge J H B, Smolley J R, Spitzer K W. The relationship between charge movements associated with I_(Ca) and I_(Na-Ca) in cardiac myocytes [J]. Science, 1990, 248(4953): 376-378.
74. Amran M S, Homma N, Hashimoto K. Pharmacology of KB-R7943: a Na~+-Ca~(2+) exchange inhibitor [J]. Cardiovasc. Drug Rev., 2003, 21(4): 255-276.
75. Amran MS, Hashimoto K, Homma N. Effects of sodium-calcium exchange inhibitors, KB-R7943 and SEA0400, on aconitine-induced arrhythmias in guinea pigs in vivo, in vitro, and in computer simulation studies [J]. J. Pharmacol. Exp. Ther., 2004, 310(1): 83-89.
76. Liu Y, Zhang SW, Liang M, et al. Effects of aconitine on [Ca~(2+)] oscillation in cultured myocytes of neonatal rats [J]. J Huazhong Univ Sci Technolog, 2008, 28(5): 499-503.
77. Harberd N, Artavanis-Tsakonas S, Berridge MJ, Bourne H, Cantley L, Downward J, et al. Cell Communication. In: Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, eds. Molecular Biology of the Cell[M]. Garland Science, 4~(th) Ed. New York, NY: Taylor & Francis Group, 2002, 831-906.
78. Harberd N, Artavanis-Tsakonas S, Berridge M J, et al. Cell Communication. In: Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, eds. Molecular Biology of the Cell. Garland Science, 4~(th) Ed. New York, NY: Taylor & Francis Group, 2002, 831-906.
79. Van Rijen HVM, Eckardt D, Degen J. et al. Slow conduction and enhanced anisotropy increase the propensity for ventricular tachyarrhythmias in adult mice with induced deletion of connexin43 [J]. Circulation, 2004, 109(8): 1048-1055.
80. Zhang Y, Kakinuma Y, Ando M, et al. Acetylcholine inhibits the hypoxia-induced reduction of connexin43 protein in rat cardiomyocytes [J]. J Pharmacol Sci, 2006, 101(3): 214-222.
81. Gutstein D E, Morley G E, Tamaddon H, et al. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43 [J]. Circ. Res., 2001, 88(3): 333-339.
82. Dhein S. Pharmacology of gap junctions in the cardiovascular system [J]. Cardiovasc Res, 2004, 62(2): 287-298.
83．王云胜，林吉进．连接蛋白43磷酸化状态与心脏缝隙连接通道功能的关系[J]．国际心血管病杂志，2007，34(4)：270-273．
84. Axelsen LN, Stahlhut M, Mohammed S, et al. Identification of ischemia-regulated phosphorylation sites in connexin43: a possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123) [J]. J Mol Cell Cardiol, 2006, 40(6): 790-798.
85. Zhang S-W, Liu Y, Huang G-Z, et al. Aconitine alters connexin43 phosphorylation status and [Ca~(2+)] oscillation patterns in cultured neonatal rat ventricular myocytes [J]. Toxicol. In Vitro, 2007, 21(8): 1476-1485.
86. Beardslee MA, Lerner DL, Tadros PN, et al. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia [J]. Circ Res, 2000, 87(8): 656-662.
87. Matsushita S, Kurihara H, Watanabe M, et al. Alterations of phosphorylation state of connexin43 during hypoxia and reoxygenation are associated with cardiac function [J]. J Histochem Cytochem, 2006, 54(3): 343-353.
2．刘良，刘艳，黄光照．389例不同年份的中毒尸检资料分析[J]．刑事技术．1999，(6)：16-18．
3．张益鹄．中国特色的法医毒理学[J]．法医学杂志．2004，20(2)：101-104．
4．熊丽娟．中药毒性理论探讨[J]．云南中医学院学报，2007，30(3)：20-23．
5．罗贯中．三国演义[M]．北京：人民文学出版社，1997，617-623．
6．符华林．我国乌头属药用植物的研究概况[J]．中药材，2004，27(2)：149-152．
7. Van Landeghem A A, De Letter E A, Lambert W E, et al. Aconitine involvement in an unusual homicide case [J]. Int. J. Legal Med., 2006, 121(3):214-219.
8．张仲景著．《金匮要略》[M]．人民卫生出版社，2004年，21-25
9．蔡建成．毒剧中药的临床应用与体会[J]．中医药学报，2004，32(4)：44-45
10．张世忠，昊博威．乌头碱与钾离子通道激动剂合用对离体大鼠心脏的正性肌力作用研究[J]．中国药理学通报，2001，17(5)：570-572．
11．王华灵，韩培秀，徐世明．乌头碱对癌症疼痛的治疗效果[J]．中国中西医结合杂志，1994，14(4)：219-223．
12. Hikino H, Takata H, Fujiwara M, et al. Mechanism of inhibitory action of Mesaconitine in acute inflammations[J]. Eur J Pharmac, 1982, (82):65-71.
13．黄光照，汪德文．法医毒理学 第3版[M]．北京：人民卫生出版社，2004，218-222．
14. Baselt RC. Disposition of Toxic Drugs and Chemicals in Man, 7th ed [M]. Biomedical publications, Foster City, 2004, 357-363.
15. Camps FE, Gradwohl's Legal Medicine, 3rd ed [M]. John Wright & Sons, Bristol, 1976.
16．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
17. Elliott SP. A case of fatal poisoning with the aconite plant: quantitative analysis in biological fluid. Sci Justice, 2002, (42) :111-115.
18．何兴元．急性乌头碱中毒56例临床分析[J]．中华医学研究杂志，2006，6(12)：1381．
19. Ravi P., Lorraine Y., Barry C., et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion. J Forensic Sci,2008, 53:491-494.
20. Lin CC, Chan TYK, Deng JF. Clinical features and management of herb-induced aconitine poisoning [J]. Ann. Emerg. Meal., 2004, 43: 574-579.
21. Moritz F, Compagnon P,Kaliszczak IG, et al. Severe acute poisoning with homemade aconitum napellus capsules: toxicokinetic and clinical data [J]. J. Toxicol-Clin. Toxic., 2005, 43: 873-876.
22．柳大勇，郭鹏毅，樊栓．乌头碱中毒致死分析1例[J]．刑事技术2007．(6)：59．
23．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
24．龚冬悔，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
25. Amran M S, Homma N, Hashimoto K. Pharmacology of KB-R7943: a Na+-Ca~(2+) exchange inhibitor [J]. Cardiovasc. Drug Rev., 2003, 21(4):255-276.
26. Amran M S, Hashimoto K, Homma N. Effects of sodium-calcium exchange inhibitors, KB-R7943 and SEA0400, on aconitine-induced arrhythmias in guinea pigs in vivo, in vitro, and in computer simulation studies [J]. J. Pharmacol. Exp. Ther., 2004.310(1): 83-89.
27. Yan LIU, Shi-Wei ZHANG, Man LIANG, Qian LIU, Liang LIU. Effects of Aconitine on [Ca~(2+)] Oscillation in Cultured Myocytes of neonatal rats. Journal of Huazhong University of Science and Technology. 2008,28(5):499-503.
28. Harberd N, Artavanis-Tsakonas S, Berridge MJ, Bourne H, Cantley L, Downward J, et al. Cell Communication. In: Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, eds. Molecular Biology of the Cell. Garland Science, 4th ed. New York, NY: Taylor & Francis Group, 2002, 831-906.
29. Fonres P., Ratel S., Lecomte D.. Pathology of arrythmogenic right ventricular cardiopathy autopsy study of forensic cases[J].J Forensic Sci, 1998,43(4):773-783.
30. James T N. Normal and abnormal consequences of apoptosis in the human heart, form postnatal morphogenesis to parocymal arrbymias [J]. Circulation, 1994,90(1):557-573.
31．雷怀成，宋道江，易建华，等．大鼠乌头碱中毒心肌细胞凋亡的研究．中国工业医学杂志．2004，17(6)：373-374．
32. Zhang S-W, Liu Y, Huang G-Z, et al. Aconitine alters connexin43 phosphorylation status and [Ca~(2+)] oscillation patterns in cultured neonatal rat ventricular myocytes [J]. Toxicol. In Vitro, 2007,21 (8): 1476-1485.
33. Axelsen LN, Stahlhut M, Mohammed S, et al. Identification of ischemia-regulated phosphorylation sites in connexin43: a possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123)[J]. J. Mol. Cell. Cardiol., 2006,40:790-798.
34. Beardslee MA, Lerner DL, Tadros PN, et al. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia [J]. Circ. Res., 2000, 87:656-662.
35. Matsushita S, Kurihara H, Watanabe M, et al. Alterations of phosphorylation state of connexin43 during hypoxia and reoxygenation are associated with cardiac function [J]. J. Histochem. Cytochem., 2006, 54: 343-353.
36. Wang SY, Wang GK. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell. Signal., 2003, 15:151-159.
1．张益鹄．中国特色的法医毒理学．法医学杂志．2004 20(2)：101-104．
2．黄光照．《法医毒理学》．第三版，2004年．
3．http://www.med66.com/new/53a273aa2009/200924qiji11135.shtml 21世纪毒理学的发展趋势．2009年2月1日．
4．鄂征主编．《组织培养和分子细胞学技术》．北京出版社，第二版，1995年．
5. Wenzel KG, Wheatley JW, Don Byrd G, et al. Effect of nicotine on cultured rat heat cells [J]. Toxicol. Appl. Pharmacol., 1970, 17: 774-785.
6. Limaye DA, Shaikh ZA. Cytotoxicity of cadmium and characteristics of its transport in cardiomyocytes [J]. Toxicol. Appl. Parmacol., 1999, 154: 59-66.
7. Acosta D, Ramos K, Cardiotoxicity of tricyclic antidepressants in primary cultures of rat myocardial cells [J]. J. Toxicol. Environ. Health, 1984, 14: 137-143.
8. Estevez MD, Wolf A, Schramm U. Effect of PSC 833, Verapamil and amiodarone on adriamycin toxicity in cultured rat cardiomyocytes [J]. Toxicol. In Vitro, 2000, 14:17-23.
9. Manabu N, Stuart WR, James EK, et al. Ouabain increases sarcoplasmic reticulum calcium release in cardiac myocytes [J]. J. Pharmacol. Exp. Ther., 2004, 308: 1181-1190.
10．章诗伟．乌头碱对培养新生大鼠心室肌细胞Connexin43蛋白磷酸化状态及其胞内[Ca~(2+)]振荡模式的影响[D]．华中科技大学同济医学院博士研究生毕业论文，2006：19-40．
11. Harary I, Farley B. In vitro studies of isolated beating heart cells[J]. Science, 1960,131 : 1674-1675.
12. Simpson N.H., Milner, A.E. and Al-Rubeai, M.. Prevention of hybridoma cell death by bcl-2 during sub optimal culture conditions[J]. Biotechnol. Bioeng, 1997,54: 1-16.
13. Yamashita N, Nishida M, Hoshida S, et al. Induction of manganese superoxide dismutase in rat cardiac myocytes increases tolerance to hypoxia 24 hours after preconditioning [J]. J. Clin. Invest., 1994, 94: 2193-2199.
14．王宁，李应东．乳鼠心肌细胞原代培养方法的改进[J]．中国心血管病研究，2007，5(12)：920-922．
15．王涛，余志斌，谢满江，等．新生大鼠心肌细胞培养技巧[J]．第四军医大学学报，2003，24(2)：92．
16．陈淳媛，孙跃女，杨作威，等．新生大鼠心肌细胞原代培养[J]．实用预防医学，2007，14(4)：983-985．
17. Ray M, Srivastava S, Maitra S, et al. The hamster heart is resistant to calcium paradox [J]. Pharmacol. Res., 2000, 41:475-481.
18. Harary I, Farley B. In vitro studies on single beating rat heart cells [J]. Cell Res. 1963, 29: 451-474.
19. Blondel B, Roijem I, Cheneval JP.Heart cells in culture: a simple method for increasing the proportion of myoblasts [J]. Exprerientia 1971, 27: 356-358.
20. Grynberg A, Athias P, Degois M. Effect of change in growth environment on cultured myocardial cells investigated in a standardized medium [J]. In Vitro Cell. Dev. Biol. 1986, 22: 44-50.
21. Hoshida S, Nishida M, Yamashita N, et al. Heme oxygenese-1 expression and its relationto oxidative stress during primary culture of cardiomyocytes [J]. J. Mol. Cell Cardiol., 1996, 28: 1845-1855.
22. Saffitz JE, Green KG, Kraft WJ, et al. Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium [J]. Am. J. Physiol. Heart Circ. Physiol., 2000, 278:H1162-H1170.
23. Zeevi-Levin N, Barac YD, Reisner Y, et al. Gap junctional remodeling by hypoxia in cultured neonatal rat ventricular myocytes [J]. Cardiovasc. Res., 2005, 66: 64-73.
24．李润琴，慕晓玲．新生大鼠心肌细胞培养技术[J]．山西医药杂志，2007，36(2)：103-105．
25. Orita H, Fukasawa M, Hirooka S, et at. Cytoprotective effects of nicorandil on hypothermic injury to immature cardiac myocytes [J]. Jpn. Circ. J., 1994, 58: 653-661.
26. Mark GE, Strasser FF. Pacemaker activity and mitosis in cultures of newborn rat heart ventricle cells [J]. Exp. Cell Res., 1966, 44: 217-233.
27. Nagy JI, Li WEI, Roy C, et al. Selective monoclonal antibody recognition and cellular localization of an unphosphorylated form of connexin43 [J]. Exp. Cell Res., 1997, 236: 127-36.
28. Ross PD, McCarl RL. Oxidation of carbohydrates and palmitate by intact cultured neonatal rat heart cells [J]. Am. J. Physiol., 1984, 246: H389-H397.
29. Simpson P, Savion S, Differentiation of rat myocytes in single cell cultures with and without proliferating non-myocardial cells [J]. Circ. Res., 1982, 50:101-116.
30. Karl S, Christoph G, Thomas F, et al. Inhibition of the ubiquitin-proteasome pathway induces differential heat-shock protein response in cardiomyocytes and renders early cardiac protection [J]. Biochem. Bioph. Res. Co., 2002, 291: 542-549.
31. Stahlhut M, Petersen JS, Hennan JK, et al. The antiarrhythmic peptide rotigaptide (ZP132) increases connexin 43 protein expression in neonatal rat ventricular cardiomyocytes [J]. Cell. Commun. Adhes., 2006, 13:21-27.
32. L(u|¨)ss H, Schmitz W, Nermann J. A proteasome inhibitor confers cardioprotection [J]. Cardiovasc. Res., 2002, 54: 140-151.
33. Marengo FD, Wang S, Wang B, Langer GA. Dependence of cardiac cell Ca~(2+) permeability on sialic acid-containing sarcolemmal gangliosides [J]. J. Mol. Cell Cardiol., 1998, 30: 127-137.
34. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limitedby extra-cellular regulated kinases 1/2-mediated induction of cyclooxygenase-2 [J]. J. Biol. Chem., 1999, 274: 5038-5046.
35. Brooks G, Poolman RA, McGill CJ, et al. Expression and activities of cyclins and cyclin-dependent kinases in developing rat ventricular myocytes [J]. J. Mol. Cell Cardiol., 1997, 29: 2261-2271.
36. Wagner DR, Combes A, McTiernan Ch, et al. Adenosine inhibits lipopoly-saccharide induced cardiac expression of tumor necrosis factor-α[J]. Circ. Res., 1998, 82: 47-56.
37. Rubin Y, Kesslericekson G, Navon G. The effect of furosemide on calcium ion concentration in myocardial-cells [J]. Cell. Calcium, 1995, 18:135-139.
38. Matos MJ, Post JA, Roelofsen B, et al. Composition and organization of sarcolemmal fatty acids in cultured neonatal rat cardiomyocytes [J]. Cell. Biol. Int. Rep., 1990, 14: 343-352.
39. Tanka M, Ito H, Adachi S, et al. Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes [J]. Circ. Res., 1994, 75: 426-433.
40. Yasui K, Kada K, Hojo M, et al. Cell-to-cell interaction prevents cell death in cultured neonatal rat ventricular myocytes [J]. Cardiovasc. Res., 2000, 48: 68-76.
41．薛庆善．体外培养的原理与技术[M]．北京：科学出版社，2001，618-636．
42．杨晓宁，田宗文，黄焕斌，等．新生大鼠心肌细胞的体外培养[J]．解剖学杂志，2005，28(3)：361-362．
43．宋学立，钱令嘉．大鼠心肌细胞的培养纯化及其在药理毒理学中的应用[J]．实用医药杂志，2002，19(12)：950-951．
1．黄光照，汪德文主编．法医毒理学[M]．第3版．北京：人民卫生出版社，2004，218-222．
2．柳大勇，郭鹏毅，樊栓良．乌头碱中毒致死分析1例[J]．刑事技术，2007，(6)：59．
3. Elliott SP.A case of fatal poisoning with the aconite plant: quantitative analysis in biological fluid[J]. Sci Justice, 2002, 42:111-115.
4. Ravi P,Lorraine Y, Barry C, et al. A Case of fatal aconitine poisoning by monkshood ingestion[J]. J Forensic Sci,2008,53:491-494.
5．苏平，刘明俊．大鼠乌头碱中毒心肌超微结构的改变[J]．西安医科大学学报．1991，12(4)：321-324．
6．张桥主编．卫生毒理学基础[M]．第三版．北京：人民卫生出版社，2001：236-240．
7．刘世新，刘艳主编．实用生物组织学技术(21世纪高等医药院校教材)[M]．北京：科学出版社，2004年3月．50-146．
8．吴景录．32例急性乌头碱中毒的临床总结．中外健康文摘·医药卫生版，2007，4(7)：54-56．
9．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
10．蔡琪琳．急性乌头碱中毒10例临床分析[J]．中国中西医结合急救杂志，2007，14(1)：59．
11．张丽娟，高顺梅．乌头碱类中毒的临床观察及护理体会[J]．黑龙江医学，2002，26(2)：139．
12．赵庆桃．乌头碱中毒45例分析[J]．中国误诊学杂志，2004，4(1)：121-122．
13．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
14．杨庆君，尹琼，万书平．乌头碱中毒致心律失常10例临床分析fJ]．中国中西医结合急救杂志，2007，14(5)：274．
15．杨维明，寿天佑．乌头碱中毒致室性心律失常16例诊治分析[J]．浙江临床医学．2006，8(10)：1072．
16．林津，徐红，徐文仪，等．重度乌头碱中毒心律失常的抢救体会[J]．内科急危重症杂志．2002，8(4)：219-220．
17．邹华．一起乌头碱引起12人食物中毒的调查[J]．中国饮食卫生与健康．2005，3(5／6)：50-51．
18．施玉樑，魏仁榆，周念辉，等．乌头碱对神经肌肉接头电活动和神经干复合电位作用的观察[J]．生理学报，1980，32(2)：135-142．
19．张宏顺．乌头类中药毒性及中毒处理[J]．药物不良反应杂志，2005，7(2)114-115．
20．卢中秋，胡国新．乌头碱急性中毒及诊治研究现状[J]．中国中西医结合急救杂志，2005，12(2)：119-121．
21. Wang S Y, Wang G K. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell Signal, 2003,15(2): 151 - 159.
22．龚冬梅，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
23. Moric E, Herbert E, Trusz-Gluza M, et al. The implications of genetic mutations in the sodium channel gene (SCN5A) [J]. Europace, 2003, 5(4): 325-334.
24．刘岩．乌头类生物碱心肌毒性作用的研究[D]．沈阳：沈阳药科大学，2007，42-49．
25. Dhein S. Pharmacology of gap junctions in the cardiovascular system [J]. Cardiovasc Res, 2004, 62: 287-298.
26. Van Veen TAB, Van Rijen HV, Opthof T. Cardiac gap junction channels modulation of expression and channel properties [J]. Cardiovasc. Res., 2001, 51: 217-229.
27. Gutstein DE, Morley GE, Tamaddon H, et al. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43 Circ. Res., 2001, 88: 333-339.
28. Kl(?)ber AG. Mechanisms of ventricular arrhythmias: a perspective [J]. J. Cardiovasc. Pharmacol., 1991, 17:S1-S8.
29. Peters NS, Coromilas J, Severs NJ, et al. Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia [J]. Circulation, 1997, 95: 988-996.
30. Zhang S-W, Liu Y, Huang G-Z, et al. Aconitine alters connexin43 phosphorylation status and [Ca~(2+)] oscillation patterns in cultured neonatal rat ventricular myocytes [J]. Toxicol. In Vitro, 2007, 21(8): 1476-1485.
1. Fairbairn DW, Olive PL , O'Neill KL. The comet assay : a comprehensive review[J]. Mutat Res , 1995, 339 (1) :37 - 59.
2．王静，万树青．彗星试验在环境毒理与农药遗传毒性研究中的应用展望[J]．生物技术通报．2003，第1期：10-12．
3. Van Landeghem A A, De Letter E A, Lambert W E, et al. Aconitine involvement in an unusual homicide case [J]. Int. J. Legal Med., 2006, 121(3):214-219.
4. Ravi P., Lorraine Y., Barry C., et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion. J Forensic Sci, 2008; 53:491-494.
5. Lin CC, Chan TYK, Deng JF. Clinical features and management of herb-induced aconitine poisoning [J].Ann. Emerg. Med., 2004, 43: 574-579.
6．柳大勇，郭鹏毅，樊栓．乌头碱中毒致死分析1例[J]．刑事技术2007年，第6期：59．
7．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
8．黄光照，汪德文．法医毒理学[M]．第3版．北京：人民卫生出版社，2004：218-222．
9．龚冬梅，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
10. Amram M S, Hashimoto K, Homma N. Effcets of sodium-calcium exchang inhibitors, KB-R7943 and SEA0400, on acontine-induced arrhythmias in guinea pigs in vivo, in vitro, and in computer simulation studies[J]. J Pharmacol Exp Ther, 2004, 310: 83-89.
11．刘艳，朱传红，邓立斌，等．毒鼠强诱导细胞DNA损伤的彗星电泳检测[J]．中国法医学杂志，2004，19(5)：278-279．
12. Konca K, Lankoff A, Banasik Anna, et al. A cross-platform public domain PC image-analysis program for the comet assay[J]. Mutat Res, 2003, 534: 15-20.
13．张家放主编．医用多元统计方法[M]。湖北：华中科技大学出版社．2002：39-59．
14．高丰，金天明，姜宁，等．细胞凋亡的研究现状与发展趋势[J]．动物医学进展，2003，24(6)：1-3．
15. Wang JYJ. DNA damage and apoptosis[J]. Cell and Differentiation. 2001,8(11):1047-1048.
16. Chris J N , Boris Z. DNA damage-induced apoptosis[J]. Oncogene (2004) 23, 2797-2808.
17. Roos WP, Kaina B. DNA damage-induced cell death by apoptosis[J].Trends Mol Med. 2006, 12(9):440-450.
18. Gabriela F. R, Manuela CR, Bj(o|¨)rn J. Characterization of DNA Damage in Yeast Apoptosis Induced by Hydrogen Peroxide, Acetic Acid, and Hyperosmotic Shock. Molecular Biology of the Cell.2006, 17 (10): 4584-4591.
19．雷怀成，宋道江，易建华，等．大鼠乌头碱中毒心肌细胞凋亡的研究[J]．中国工业医学杂志．2004，17(6)：373-374．
20. Nakayama T, Kaneko M, Kodama M, et al. Volatile gas components contribute to cigarette smoke induced DNA single strand breaks in cultured humancells[J]. Agric Biol Chem 1986;50:3219-3220.
21. Hruszkewycz AM and Bergtold DS. The 8-hydroxyguanine content of isolated mitochondria increases with lipid peroxidation[J]. Mutat Res. 1990, 244, 123-128.
22．刘晓麒，曹恩华．脂质过氧化引起的DNA损伤研究进展[J]．生物化学与生物物理进展，1994，21(3)：218-222．
23．李雨民，杨凤桐．DNA损伤修复与细胞凋亡[J]．国外医学-放射医学核医学分册．1999，23(3)：112-115．
24. Lu YC, Huang CC, Huang C J, et al. Effects of Antrodia camphorata on viability, apoptosis, [Ca2+] i, and MAPKs phosphorylation in MG63 human osteosarcoma cells[J].Drug Development research. 2007, 68(2):71-78.
25. Wang X , Chen S, Ma G , et al. Involvement of proinflammatory factors, apoptosis , caspase-3 activation and Ca2+disturbance in microglia activation-mediated dopaminergic cell degeneration[J]. Mech. Ageing Dev. , 2005, 126: 1241-1254.
26．王华，张天一，杨扬，等．三氧化二砷诱导黑色素瘤B16细胞凋亡机制[J]．江苏医药，2006，32(12)：1128-1129．
27．许惠玉，陈志伟，牛建昭，等．赤芍总苷诱导K562细胞凋亡及对线粒体膜电位和Ca~(2+)的影响[J]．中国组织工程研究与临床康复，2008，12(16)：3123-3126．
28．付敏．乌头碱致心律失常的细胞分子机制的实验研究[D]．北京中医药大学博士研究生学位论文．2007：56-72．
29. Eigel BN, Gursahani H, Hadley RW. Na+/Ca2+ exchanger plays a key role in inducing apoptosis after hypoxia in cultured guinea pig ventricular myocytes[J]. Am J Physiol Heart Circ Physiol, 2004, 287:1466-1475.
30. Rodriguez Tarduchy G, Sahuquillo AG, Alarcon B et al.Apoptosis but not other activation events is inhibited by a mutation in the transmembrane domain of T cell receptor beta that impairs CD3zeta association[J].J Biol Chem, 1996, 271(48): 30417-30425.
31. Yan Liu, Shi-Wei Zhang, Man Liang, et al. Effects of Aconitine on [Ca2+] Oscillation in Cultured Myocytes of neonatal rats[J]. J Huazhong Univ Sci Technolog, 2008, 28(5): 499-503.
32．肖琳，王松，王凡．应用彗星实验检测细胞DNA损伤的原理与方法．四川解剖学杂志，2005，12(1)：39-42．
33. Lin SM, Yang MH. Arsenic, selenium, and zinc in patients with Blackfoot disease[J]. Biol Trace Elem Res. 1988, 15:213-221.
34．邢彩虹，李桂兰，纪之莹，等．单细胞凝胶电泳图像分析软件的比较．毒理学杂志．2005，19(2)：141-143．
35．张建平．单细胞凝胶电泳技术及其应用．国外医学遗传学分册，1997，20(5)：231-234．
36. Lee E, Oh E, Lee J , et al. Use of the tail moment of the lymphocytes to evaluate DNA damage in human biomoitoring studies[J]. Toxicol Sci, 2004, 81(1): 121 - 132.
37. Singh R, Sram RJ, Binkova B, et al. The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans[J]. Mutat Res, 2007, 620: 83- 92.
38. Cebulska WA, Binkova B, Sram RJ, et al. Repair competence assay in studies of the influence of environmental exposure to c-PAHs on individual susceptibility to induction of DNA damage[J]. Mutat Res, 2007, 620: 155- 164.
39. Ostling O, Johanson, KJ. Microelectrophoretic study of radiation-induced DNA damage in individual mammalian cells[J].Biochem Biophys Res Commun. 1984,123(2):291-298.
40. Singh R, Sram RJ, Binkova B, et al. The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans[J]. Mutat Res, 2007, 620: 83- 92.
1. Sohl G.,Willecke K. Gap junctions and the connexin protein family[J]. Cardiovasc.Res, 2004,62:228-232.
2. Dhein S, Polontchouk L, Salameh JA.Pharmacological modulation and differential regulation of the cardiac gap junction proteins connexin 43 and connexin 40[J]. Biology of the Cell ,2002, 94: 409-422.
3. Dupont E.Matsushita T.Kaba RA Altered connexin expression in human congestive heart failure[J]. J Mol Cell Cardiol. 2001,33:359-371.
4．李霞，张存泰，王琳，等．心力衰竭时CX43的改变及其作用[J]．中国心脏起搏与心电生理杂志，2005，19(5)：404-406．
5．苏德淳，常志文，范书英．缝隙连接蛋白43在缺血性心律失常中的作用[J]．首都医科大学学报，2006，27：235-238．
6. Schu lz R,Heusch G. Connexin 43 and ischem icprecond ition ing .Card iovasc Res, 2004,62, 62:335-344.
7. Giepmans BN. Gap junctions and connexin-interacting proteins .Cardiovasc Res, 2004,622, 62(2) :233-245.
8. Salameh A. Life cycle of connexins:regulation ofconnexin sythesis and degradation[J] .Adv Cardiol. 2006,42, 42:57-70.
9. Saez JC,Retamal MA,Basilio D,et al. Connexin-based gap junction hemichannels:gating mechanisms[J] .Biochim Biophys Acta. 2005,1711, 1711:215-224.7. Baselt RC. Disposition of Toxic Drugs and Chemicals in Man, 7th ed [M]. Biomedical publications, Foster City, 2004, 357-363.
10. Lin H,Ogawa K,Imanaga I,et al. Alterations ofcormexin 43 in the diabetic rat heart[J] .Adv Cardiol. 2006,42, 42:243-254.
11．王荣；张存泰；王琳．缝隙连接蛋白Cx43的磷酸化对缝隙连接通讯的调控[J]．中国心血管杂志，2008，
12．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
13．赵庆桃．乌头碱中毒45例分析[J]．中国误诊学杂志，2004，4(1)：121-122．
14．蔡琪琳．急性乌头碱中毒10例临床分析[J]．中国中西医结合急救杂志，2007，14 (1)：59．
15．张丽娟，高顺梅．乌头碱类中毒的临床观察及护理体会[J]．黑龙江医学，2002，26(2)：139．
16．章诗伟，任杰林，张黎，等．乌头碱对培养新生心室肌细胞Connexin 43蛋白磷酸化的影响[J]．中国法医学杂志，2008，23(2)：29-32．
17. Beardslee MA, Lerner DL, Tadros PN, et al. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia [J]. Circ. Res., 2000, 87:656-662.
18. Turner MS, Haywood GA, Andreka P, et al. Reversible connexin 43 dephosphorylation during hypoxia and reoxygenation is linked to cellular ATP levels [J]. Circ. Res., 2004, 95: 726-733.
19. M(u|¨)ller A, Gottwald M, Tudyka T, et al. Increase in gap junction conductance by an antiarrhythmic peptide [J]. Eur. J. Pharmacol., 1997, 327:65-72
20. M(u|¨)ller A, Schaefer T, Linde W, et al. Actions of the antiarrhythmic peptide AAP10 on cellular coupling [J]. Naunyn. Schmiedebergs. Arch. Pharmacol., 1997, 356: 76-82.
21. Dhein S, Weng S, Polontehouk L, et al. Pharmacological modification of gap junctional coupling by antiarrhythmic peptides. Role of PKC [J]. Naunyn. Schmiedebergs. Arch. Pharmacol., 2001, 363:R99 [Suppl.].
22. Weng S, Lauven M, Schaefer T, et al. Pharmacological modulation of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation [J]. FASEB J. 2002, 16: 1114-1116.
23. Axelsen LN, Stahlhut M, Mohammed S, et al. Identification of ischemia-regulated phosphorylation sites in connexin43: a possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123) [J]. J. Mol. Cell. Cardiol., 2006, 40: 790-798.
24. Stahlhut M, Petersen JS, Hennan JK, et al. The antiarrhythmic peptide rotigaptide (ZP132) increases connexin 43 protein expression in neonatal rat ventricular cardiomyocytes [J]. Cell. Commun. Adhes., 2006, 13:21-27.
25. Matsushita S, Kurihara H, Watanabe M, et al. Alterations of phosphorylation state of connexin43 during hypoxia and reoxygenation are associated with cardiac function [J]. J. Histochem. Cytochem., 2006, 54: 343-353.
26. Dhein S, Weng S, Polontehouk L, et al. Pharmacological modification of gap junctional coupling by antiarrhythmic peptides. Role of PKC [J]. Naunyn. Schmiedebergs. Arch. Pharmacol., 2001, 363:R99 [Suppl.].
27. Weng S, Lauven M, Schaefer T, et al. Pharmacological modulation of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation [J]. FASEB J. 2002, 16: 1114-1116.
28. Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication [J]. Int. J. Biochem. Cell Biol., 2004, 36:1171-1186.
29．李喜涛，许文灿．连接蛋白43磷酸化状态与缝隙连接通道功能的关系．汕头大学医学院学报，2008，21(3)：185-188．
30．庄燕燕，唐雪莲．间隙连接及其磷酸化调节．国际病理科学与临床杂志．2006，26(5)：444-447．
31. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses [J]. Am. J. Physiol.-Lung C. 2000, 279: L429-L438.
32. Joell L S, Paul D L. Connexin phosphorylation as a regur latory event linked to gap junction channel assembly[J]. Biochimica et Biophysica Acta, 2005,1711(2):154-163.
33. EK-Vttop J F, King T J, Heyman N S, et al. Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation[J]. Circ Res, 2006.98:1498-1505.
34. Zeevi-Levin N, Barac YD, Reisner Y, et al. Gap junctional remodeling by hypoxia in cultured neonatal rat ventricular myocytes [J]. Cardiovasc. Res., 2005, 66: 64-73.
35. Saffitz JE, Green KG, Kraft WJ, et al. Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium [J]. Am. J. Physiol. Heart Circ. Physiol., 2000, 278:H1162-H1170.
36．张家放主编．医用多元统计方法[M]。湖北：华中科技大学出版社．2002：39-59．
37. Lampe PD, Lan AF.The effects of connexin phosphorylation on gap junctional communication[J]. Biochem Cell Biol,2004,36:1171-1186.
38. Beardslee MA, Lerner DL, Tadros PN, et al. Dephosphorylation and intracellular redistribution of ventricular connexin 43 during electrical uncoupoing induced by ischemial[J].Circ Res, 2000,87:656-662.
39. Guerrero PA, Schuessler RB, Davis LM, et al. Slow ventricular conduction in mice heterozygous for a connexin 43 null mutation[J]. J Clin Invest, 1997,99:1991-1998.
40. Thomas SA, Schuessler RB, Berul CI, et al. Disparate effects of deficient expression of connexin 43 on atrial and ventricular conduction : evidence for chamber-specific molecular determinations of conduction [J]. circulation, 1998, 97:686-691.
41. Dempsey EC, Newton AC, Mochly-Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses. Am. J. Physiol.-Lung C, 2000, 279:L429-L438.
42. Parekh DB, Ziegler W, Parker PJ. Muliple pathways control protein kinase C phosphorylation. EMBO J., 2000,19: 496-503.
43. Bornancin F, Parker PJ. Phosphorylation of protein kinase C-α on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state. J. Biol. Chem., 1997, 272: 3544-3549.
44. Weng S, Lauven M, Schaefer T, et al. Pharmacological modulation of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation [J]. FASEB J. 2002,16: 1114-1116.
45. Dhein S, Weng S, Grover R, et al. Protein kinase C alpha mediates the effect of antiarrhythmic peptide on gap junction conductance [J]. Cell Commun. Adhes., 2001,8:257-264.
46. Van Veen TAB, Van Rijen HV, Opthof T. Cardiac gap junction channels modulation of expression and channel properties [J]. Cardiovasc. Res., 2001, 51: 217-229.
47. Kumar NM, Gilula NB. The gap junction communication channel [J]. Cell, 1996, 84: 381-388.
48. Dhein S. Pharmacology of gap junctions in the cardiovascular system [J]. Cardiovasc Res, 2004, 62: 287-298.
49. Axelsen LN, Stahlhut M, Mohammed S, et al. Identification of ischemia-regulated phosphorylation sites in connexin43: a possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123) [J]. J. Mol. Cell. Cardiol., 2006, 40: 790-798.
50. Matsushita S, Kurihara H, Watanabe M, et al. Alterations of phosphorylation state of connexin43 during hypoxia and reoxygenation are associated with cardiac function [J]. J. Histochem. Cytochem., 2006, 54: 343-353.
51. Muller A, Gottwald M, Tudyka T, et al. Increase in gap junction conductance by an antiarrhythmic peptide [J]. Eur. J. Pharmacol., 1997, 327: 65-72.
52. Muller A, Schaefer T, Linde W, et al. Actions of the antiarrhythmic peptide AAP10 on cellular coupling [J]. Naunyn. Schmiedebergs. Arch. Pharmacol., 1997,356: 76-82.
53．王荣，张存泰，阮燕菲，等大鼠心肌急性缺氧时缝隙连接蛋白43磷酸化水平的改变及心律失常肽10对其影响[J]．临床心血管疾病杂志．2007，23(5)：375-377．
54. TenBroek EM, Lampe PD, Solan JL, et al. Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP[J]. J Cell Biol,2001,155:1307-1318.
55. Lampe PD. Analyzing phorbol ester effects on gap junction communication : A dramatic inhibition of assembly[J]. J Cell Biol, 1994,127:1895-1905.
56. Lin H, Ogawa K, Imanaga I, et al. Alteration of connexin43 in the diabetic rat heart[J]. Adc Cardiol, 2006,42:243-254.
57. Wagner LM, Saleh SM, Boyle DJ, et al. Effects of protein kinase Cgammma on gap junction disassembly in lens epithelial cellsand retinal cells in culture[J]. Mol Vis,2002,8:59-66.
58. Cruciani V, Sanner T, Mikalsen SO. Pharmacological evidence for system-depenmant involvement of protein kinase C isoenzymes in phorbol ester-suppressed gap junctional communication[J]. Carcino genesis, 200122:221-231.
59. Wang S Y, Wang G K. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell. Signal., 2003, 15(2): 151-159.
60．龚冬梅，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
61. Moric E, Herbert E, Trusz-Gluza M, et al. The implications of genetic mutations in the sodium channel gene (SCN5A) [J]. Europace, 2003, 5(4): 325-334.
62．刘岩．乌头类生物碱心肌毒性作用的研究[D]；沈阳药科大学；2007年，35-48．
63．苏平，刘明俊．大鼠乌头碱中毒心肌超微结构的改变[J]．西安医科大学学报．1991，12(4)：321-324．
64．李宏，刘明俊，陈念祖．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅰ)-线粒体细胞色素C氧化酶[J]．中国法医学杂志，1988，3(4)：199-201．
65．李宏，陈念祖，刘明俊．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅱ)-线粒体琥珀酸脱氢酶(SDH)的定位[J]．中国法医学杂志，1988，3(4)：202-205．
66．梁强荣，刘明俊，胡炳蔚．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅲ)-线粒体NADHD的组织化学和细胞化学研究[J]．中国法医学杂志，1991，6(2)：84-86．
67．梁强荣，刘明俊，胡炳蔚．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅳ)-乳酸脱氢酶的组织化学和细胞化学定位[J]．中国法医学杂志，1991，6(3)：140-141．
68. Yan LIU, Shi-Wei ZHANG, Man LIANG; Qian LIU, Liang LIU. Effects of Aconitine on Ca~(2+)] Oscillation in Cultured Myocytes of neonatal rats. Journal of Huazhong University of Science and Technology. 2008,28(5):499-503.
1. Berridge MJ, Bootman MD, Roderick HL. Calcium signaling:dynamics, homeostains and remodeling [J]. MOL Cell Biol. 2003, 4:517-529.
2. Anderson ME. Calmodulin and the philosopher's stone: changing Ca~(2+) into the arrhythmias[J].J Cardiovase Electrophysiol. 2002, 13:195-197.
3. NiuH L, Pan ZW, Zhu JX, eta.l Dihydromyricetin exerted its va-soconstrictive effects by increasing intracellular Ca~(2+) in isolated canine carotid artery[J].Chin Pharmacol Bull, 2007, 23 (11) :1437-40.
4．张高小，王玲，李超，等．细胞内钙在脑心综合征心律失常中作用研究．中国药理学通报．2008，24(12)：1607-1610．
5．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
6. Elliott SP. A case of fatal poisoning with the aconite plant: quantitative analysis in biological fluid. Sci Justice, 2002;42:111-115.
7．何兴元．急性乌头碱中毒56例临床分析[J]．中华医学研究杂志，2006，6(12)：1381．
8. Ravi P., Lorraine Y., Barry C., et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion. J Forensic Sci, 2008; 53:491-494.
9. Yan Liu, Shi-Wei Zhang, Man Liang, et al. Effects of Aconitine on [Ca~(2+)] Oscillation in Cultured Myocytes of neonatal rats[J]. J Huazhong Univ Sci Technolog, 2008, 28 (5) :499-503.
10．柯俊，张存泰，马业新，等．钙调蛋白激酶Ⅱ抑制剂对心肌肥厚兔室性心律失常的影响．中华心血管病杂志．2007，35(1)：33-35．
11. Marx SO, Gaburjakova J, Gaburjakova M, Henrikson C, Ondrias K, Marks AR. Coupled gating between cardiac calcium release channels(ryanodine receptors) [J]. Circ Res 2001, 88: 1151-1158.
12. Marx SO, Ondrias K, Marks AR. Coupled gating between individual skeletal muscle calcium release channels ( ryanodine receptors ). Science 1998, 281:818-821
13. Wu X D, Dai D Z, Zhang QP, et al. Proparnolol and vera-pamil inhibit Mrna expression of RyR2 and SERCA in L-thyroxin-induced rat ventricular hypertrophy[J].Acta Pharmacol Sin. 2004, 25 (3) : 347-351.
14. Wehrens XH, Marks AR. Altered function and regulation of cardiac ryanodine receptors in cardiac disease[J].Trends Biochem Sci, 2003, 28 (12) : 671-678.
15. Wehrens X H, Lehnart S E, Huang F, et al. FKBP12·6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death[J].Cell, 2003, 113 (7) : 829-840.
16．朱传红．毒鼠强中毒的法医分子毒理学研究[D]．华中科技大学同济医学院博士研究生学位论文，2005：91-106．
17．付敏．乌头碱致心律失常的细胞分子机制的实验研究[D]．北京中医药大学博士研究生学位论文，2007：97-120．
18．刘晓宇．Wnt对Connexin43表达及其功能的影响[D]．复旦大学博士研究生学位论文，2007：38-55
19. Glover G, Mitchell J. An Introduction To Biostatistics[M]. McGraw-Hill Companies, Inc. 2001: 296.
20．张家放主编．医用多元统计方法[M]。湖北：华中科技大学出版社．2002：39-59
21．崔婕，薛绍白．胞内钙稳态调节[J]．细胞生物学杂志，1995；17(3)；97-102．
22．祝宝华，奚群英，刘晨，等．细胞膜钠钙交换蛋白在大鼠心肌缺血预适应及腺苷诱导预适应中的作用及其信号转导[J]．中华心血管病杂志．2006，34(4)：
23. Hilgemann D W, Nicoll D A, Philipson K D. Change movement during Na~+ translocation by native and cloned cardiac Na~+/Ca~(2+) exchanger [J]. Nature, 1991, 352 (6337) :715-718.
24. Bridge J H B, Smolley J R, Spitzer K W. The relationship between charge movements associated with ICa and INa-Ca in cardiac myocytes [J]. Science, 1990, 248 (4953) :376-378.
25. Xander H, Andrew R. Altered function and regulation ofcardic ryanodine receptor in cardic disease[J]. Trends Bio Sci 2003, 28 (12) : 671-67.
26. Franzini-Armstrong C, ProtastF, RameshV. Shape size and distribution of Ca~(2+) release units and couplons in skeletal and cardiacmuscles[J]. Biophys J, 1999, 77: 1528-1539
27. MeissnerG. Ryanodine receptor /Ca~(2+) release channels and their regulation by endogenous effectors[J]. AnnuRev Physiol 1994, 56: 485.
28. WilliamsA, WestD, Sitsapesan R. Light at the end of thecalcium release channel tunne:1 Structures and mechanisms involved in ion translocation in ryanodine receptor channels[J]. Q Rev Biophys, 2001, 34: 61.
29. Xander H, Andrew R. Altered function and regulation of cardic ryanodine receptor in cardic disease[ J]. Trends Bio Sci , 2003, 28 (12) : 671-67.
30. Clark M, Leo T W, Maelcrman DH.Functional consequences of alterations to polar amino acids located in the mmsmembrane domain of the Ca2 (+) -ATPase of sareoplasmic reticulum[J]J Bioehem, 1990; 265 (11) : 6262-6267.
31．蒋艳伟，刘尔亮，朱少华，等．兰尼碱受体及其法医学意义[J]．中国法医学杂志．2008，23(1)：38-40．
32．李江，胡申江，赵晓燕，等．腺相关病毒载体介导的PLB基因反义RNA对大鼠心肌细胞肌浆网Ca~(2+)-ATPase活性和[Ca~(2+)]i的作用[J]．中国病理生理杂志．2006，22(1)：39-42．
33．孙祝美．心肌细胞与钙离子调控[J]．广州医药．2008，39(3)：1-3．
34．郑霞，孙坚，胡申江，等．缺血／再灌注心肌肌浆网肌钙调控蛋白mRNA表达的变化[J]．中国应用生理学杂志．2006，22(2)：142-146．
35. Matolweni L O, Bardien S, Rebello G, et al. Arrhythmogenic right ventricular cardiomyopathy type 6 (ARVC6) : sup-port for the locus assignment , narrowing of the criticalregion and mutations creening of three candidate genes [J].BMC Med Genet, 2006, 28 (7) :29.
36. Akao Y, Nakagawa Y, AkiyamaK. Arsenic trioxide induces apoptosis in neuroblastoma cell lines through the activation of caspase-3 in vitro [J]. FEBS Lett, 1999, 455 (1-2) :59-62.
37. Yung HC, Man KH, Chong HR, et al. Induction of apoptotic cell death by mycelium extracts of Phellinus linteus in human neuroblastoma cells[J]. International Journal Of Molecular Medicine. 2004, 14: 227-232.
38. Ananth C., Thameem-Dheen S, Gopalakrishnakone P, et al. Domoic acid-induced neuronal damage in the rat hippocampus: Changes in apoptosis related genes (Bcl-2, Bax, caspase-3 ) and microglial response[J]. Journal of Neuroscience Research.2001, 66 (2) :177-190.
39. He CH, Waxman AB, Lee CG, et al. Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury[J]. J Clin Invest. 2005, 115:1039-1048.
40. Jayanthi S, Deng XL, Noailles P H, et al. Methamphetamine induces neuronal apoptosis via cross-talks between endoplasmic reticulum and mitochondria-dependent death cascades[J]. The FASEB Journal. 2004, 18:238-251.
41. Ananth, Dheen ST, Gopalakrishnakone P, et al. Distribution of NADPH-diaphorase and expression of nNOS, N-methyl-D-aspartate receptor (NMDAR1) and non-NMDA glutamate receptor (GlutR2) genes in the neurons of the hippocampus after domoic acid-induced lesions in adult rats[J]. Hippocampus. 2003, 13 (2): 260-272.
42．郭景元，李伯龄主编．中国刑事技术大全法医物证学分册[M]．北京：中国人民公安大学出版社出版．2002，335-355．
1．张益鹄．中国特色的法医毒理学[J]．法医学杂志，2004，20(2)：101-104．
2．熊丽娟．中药毒性理论探讨[J]．云南中医学院学报，2007，30(3)：20-23．
3．顾清光．神农本草经[M]．学苑出版社，2007：27．
4．张仲景．金匮要略[M]．人民卫生出版社，2004：21-25．
5．蔡建成．剧毒中药的临床应用与体会[J]．中医药学报，2004，32(4)：44-45．
6．云南省卫生厅．关于食用添加乌头碱类药物中毒的预警公告[EB／OL]．http://www. yn.chinanews.com.cn/news/html/tingjudongtai/shengweishengting/20081117/76000.html, 2008-11-17.
7．付静．食用草乌谨防乌头碱中毒[N]．保健时报，2009-2-5(002)．
8．黄光照，汪德文．法医毒理学(第3版)[M]．北京：人民卫生出版社，2004：218-222．
9．苏建宏，余成敏．68例乌头碱中毒的心电图分析[J]．中国医疗前沿，2009，4(3)：104．
10. Baselt RC. Disposition of Toxic Drugs and Chemicals in Man, 7~(th) Ed [M].Biomedical publications, Foster City, 2004, 357-363.
11. Camps FE. Gradwohl's Legal Medicine, 3~(rd) Ed [M]. John Wright & Sons, Bristol, 1976, 215-236.
12．陈安宝，陈嘉勇，梁道明，等．急性中毒2658例流行病学分析[J]．中国急救医学，2008，28(5)：409-411．
13. Van Landeghem AA, De Letter EA, Lambert WE, et al. Aconitine involvement in an unusual homicide case [J]. Int. J. Legal Med., 2006, 121(3): 214-219.
14. Ravi P., Lorraine Y., Barry C., et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion [J]. J Forensic Sci, 2008, 53: 491-494.
15. Lin CC, Chan TYK, Deng JF. Clinical features and management of herb-induced aconitine poisoning [J]. Ann. Emerg. Med., 2004, 43: 574-579.
16．柳大勇，郭鹏毅，樊栓．乌头碱中毒致死分析1例[J]．刑事技术，2007，6：59．
17．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
1．黄光照，汪德文．法医毒理学[M]．第3版．北京：人民卫生出版社，2004，218-222．
2. Elliott SP.A case of fatal poisoning with the aconite plant: quantitative analysis in biological fluid [J]. Sci Justice, 2002, 42:111-115.
3. Pullela R, Young L, Gallagher B, et al. A Case of Fatal Aconitine Poisoning by Monkshood Ingestion [J]. J Forensic Sci, 2008, 53: 491-494.
4．何亚磊，李慧娟．乌头碱中毒197例临床分析[J]．中华现代内科学杂志，2005，2(5)：444-445．
5. Mori A, Mukaida M, Ishiyama I, et al. Homicidal poisoning by aconite: report of a case from the viewpoint of clinical forensic medicine [J]. Nihon Hoigaku Zasshi, 1990, 44(4): 352-357.
6．张益鹄．中国特色的法医毒理学[J]．法医杂志学，2004，20(2)：101-104．
7．符华林．我国乌头属药用植物的研究概况[J]．中药材，2004，27(2)：149-152．
8. Van Landeghem AA, De Letter EA, Lambert WE, et al. Aconitine involvement in an unusual homicide case [J]. Int J Legal Med., 2006, 121(3): 214-219.
9．熊丽娟．中药毒性理论探讨[J]．云南中医学院学报，2007，30(3)：20-23．
10．张仲景．金匮要略[M]．北京：人民卫生出版社，2004，
11．蔡建成．毒剧中药的临床应用与体会[J]．中医药学报，2004，32(4)：44-45．
12．王华灵，韩培秀，徐世明．乌头碱对癌症疼痛的治疗效果[J]．中国中西医结合杂志，1994，14(4)：219-223．
13．张世忠，吴博威．乌头碱与钾离子通道激动剂合用对离体大鼠心脏的正性肌力作用研究[J]．中国药理学通报，2001，17(5)：570-572．
14. Hikino H, Takata H, Fujiwara M, et al. Mechanism of inhibitory action of Mesaconitine in acute inflammations [J]. Eur J Pharmacol, 1982, 82(1-2): 65-71.
15．柳大勇，郭鹏毅，樊栓．乌头碱中毒致死分析1例[J]．刑事技术，2007，31(6)：59．
16．谷振勇，丛斌，刘汝俊．乌头碱中毒尸检1例[J]．法医学杂志，1999，15(1)：45．
17．吕桂玲，张一民，宋兴芳，等．653例含乌头碱类药物致不良反应文献分析[J]．中国药房，2007，18(5)：374-376．
18．赵庆桃．乌头碱中毒45例分析[J]．中国误诊学杂志，2004，4(1)：121-122．
19．阮海林，陆宁．乌头碱中毒的特点及治疗(附25例临床分析)[J]．广西医科大学学报，2000，17(1)：160．
20．朱永福，宋江萍，周永丽．乌头碱类中药中毒致心律失常22例[J]．中国危重病急救医学，2000，12(3)：136．
21. Yeih DF, Chiang FT, Huang SK, et al. Successful treatment of aconitine induced life threatening ventricular tachyarrhythmia with amiodarone [J]. Heart, 2000, 84(4): E8.
22．张丽娟，高顺梅．乌头碱类中毒的临床观察及护理体会[J]．黑龙江医学，2002，26(2)：139．
23．吴景录．32例急性乌头碱中毒的临床总结．急性乌头碱中毒的临床总结[J]．中外健康文摘(医药月刊)，2007，4(7)：54-56．
24．张丽，李晔．乌头碱中毒致精神障碍2例[J]．包头医学院学报，2002，18(3)：245．
25. Ohuchi S, Izumoto H, Kamata J, et al. A case of aconitine poisoning saved with cardiopulmonary bypass [J]. Kyobu-Geka, 2000, 53(7): 541.
26．徐红，林津．重度乌头碱中毒致血红蛋白尿2例[J]．内科急危重症杂志，2002，8(3)：12．
27．赵初环，卢中秋，黄唯佳，等．血液净化治疗急性乌头碱中毒[J]．中华内科杂志，2001，40(7)：50．
28．苏平，刘明俊．大鼠乌头碱中毒心肌超微结构的改变[J]．西安医科大学学报，1991，12(4)：321-324．
29. Skrupskii V A, Plaksin S E. Changes in the fatty acid composition of the phospholipids in the internal organs of rats during the modeling of aconitine arrhythmia [J]. Eksp. Klin. Farmakol., 1994, 57(4): 53-55.
30．李宏，刘明俊，陈念祖．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅰ)-线粒体细胞色素C氧化酶[J]．中国法医学杂志，1988，3(4)：199-201．
31．李宏，陈念祖，刘明俊．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅱ)-线粒体琥珀酸脱氢酶(SDH)的定位[J]．中国法医学杂志，1988，3(4)：202-205．
32．梁强荣，刘明俊，胡炳蔚．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅲ)-线粒体NADHD的组织化学和细胞化学研究[J]．中国法医学杂志，1991，6(2)：84-86．
33．梁强荣，刘明俊，胡炳蔚．大鼠乌头碱中毒心肌酶的细胞化学研究(Ⅳ)-乳酸脱氢酶的组织化学和细胞化学定位[J]．中国法医学杂志，1991，6(3)：140-141．
34．陈啸梅．组织化学手册[M]．第1版．北京：人民卫生出版社．1982，220-222．
35．陈忆九，乌仁，黄晓华．心脏性猝死心肌细胞凋亡研究[J]．法医学杂志，1999，15(3)：133-134．
36. Fonres P, Ratel S, Lecomte D. Pathology of arrythmogenicri ghtve ntricular cardiopathy autopsy study of forensic cases [J]. J Forensic Sci, 1998, 43(4): 773-783.
37. James TN. Normal and abnormal consequences of apoptosis in the human heart. Form postnatal morphogenesis to paroxymal arrhythmias [J]. Circulation, 1994, 90(1): 557-573.
38．雷怀成，宋道江，易建华，等．大鼠乌头碱中毒心肌细胞凋亡的研究[J]．中国工业医学杂志，2004，17(6)：373-374．
39. Wang SY, Wang GK. Voltage-gated odium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell Signal, 2003, 15(2): 151-159.
40．龚冬梅，单宏丽，周宇宏，等．哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点[J]．药学学报，2004，39(5)：328-332．
41. Moric E, Herbert E, Trusz-Gluza M, et al. The implications of genetic mutations in the sodium channel gene (SCN5A) [J]. Europace, 2003, 5(4): 325-334.
42. Gellens ME, George AL Jr, ChenLQ, et al. Primary strueture and funetional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel [J]. Proc Natl Acad Sci U S A, 1992, 89(2): 554-558.
43. George AL, Varkony TA, et al. Assignment of the human heart tetrodotoxin resistant voltage-dependent Na channel α-subunit gene (SCN5A) to band 3p21 [J]. Cytogenet Cell Genet, 1995, 68(1-2): 67-70.
44. Wang Q, Shen JX, Splawki I, et al. SCN5A mutations associated with an inherited cardiac arrhythmia, Long QT syndrome [J]. Cell, 1995, 80(5): 805-911.
45．刘岩．乌头类生物碱心肌毒性作用的研究[D]．沈阳药科大学，2007，35-48．
46. Benitah JP, Gomez AM, Faueonnier J, et al. Voltage-gated Ca~(2+) currents in the human pathophysiologic heart: a review [J]. Basic Res Cardiol, 2002, 97 Suppl 1: 111-18.
47．于雷．心肌离子通道的研究进展[J]．新血管病学进展，2006，27(4)：413-416．
48. Patterson E, Lazzara R, Szabo B, et al. Sodium-Calcium Exchange Initiated by the Ca2+ Transient: An Arrhythmia Trigger within Pulmonary Veins [J]. J Am Coll Cardiol, 2006, 47(6): 1196-1206.
49．陈龙，马骋，蔡宝昌，等．乌头碱对大鼠心肌细胞钙通道阻滞作用的单通道分析[J]．药学学报，1995，30(3)：168-171．
50．吴晓冬，戴德哉．雷诺定受体与心律失常[J]．东南大学学报(医学版)，2005，24(1)：55．
51. Wehrens XH, Lehnart SE, Huang F, et al. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death [J]. Cell, 2003, 113(7): 829-840.
52. Priori SG, Napolitano C, Tiso N, et al. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia [J]. Circulation, 2000, 103(2): 196-200.
53. Tiso N, Stephan DA, Nava A, et al. Identification of mutations in the cardiac ryanodine receptor gene in families afected with arrhythmogenic right ventricular cardiomyopathy type 2(ARVD2) [J]. Hum Mol Genet, 2001, 10(3): 189-194.
54. Laitinen PJ, Brown KM, Piippo K, et al. Mutations of the cardiac ryanodine receptor gene in familial polymorphic ventricular tachycardia [J]. Circulation, 2001, 103(4): 485-490.
55. Sipido KR, Volders PG, de Groot SH, et al. Enhanced Ca (2+)release and Na/Ca exchange activity in hypertrophied canine ventricular myocytes: potential link between contractile adaptation and arrhythmogenesis [J]. Circulation, 2000, 102(17): 2137-2144.
56. Wehrens XH, Lehnart SE, Reiken SR, et al. Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2 [J]. Science, 2004, 304(5668): 292-296.
57．杨琳，黄诒焯．心肌细胞离子通道与心律失常[J]．中国心脏起搏与心电生理杂志，2003，17(2)：85．
58．付敏．乌头碱致心律失常的细胞分子机制的实验研究[D]．北京中医药大学，2007．
59．郭佳．钾离子通道结构的新发现[J]．河南医学研究，2006，15(2)：189-192．
60. Brown AM. Drugs, hERG and sudden death [J]. Cell Calcium, 2004, 35(6):543-547.
61．夏敏，陈义汉．心脏钾离子通道相互作用的研究进展[J]．东南大学学报(医学版)，2004，23(4)：278-281．
62．刘水平，陈玉川．心脏钾离子通道的电生理和分子多样性[J]．湖北化民族学院学报：医学版，2001，18(2)：38-40．
63. Bosch RF, Zeng X, Grammer JB, et al. Ionic mechanisms of electrical remodeling in human atrial fibrillation [J]. Cardiovasc Res, 1999, 44(1): 121-131.
64. Karle CA, Zitron E, Zhang W, et al. Human carfliac inwardly-rectifying K~+ channel kir(2.1b) is inhibited by direct protein kinase dependent regulation in human isolated cardiomyocytes and in an expression system [J]. Circulation, 2002, 106 (12): 1493-1499.
65. Wickman KD, Iniguwz-Lluhi JA, Davenport PA, et al. Reconr binant G-protein β_γ-subunits activate the muscarinic- gated atrial potassium channel [J]. Nature, 1994, 368(6468): 255-257.
66. Liu Y, Xu CQ, Jiao JD, et al. M_3-R/I_K(M3)--a new target of antiarrhythmic agents [J]. Yao Xue Xue Bao, 2005, 40(1): 8-12.
67. Grover GJ, Garlid KD. ATP-Sensitive potassium channels: a review of their cardioprotective pharmacology [J]. J Mol Cell Cardiol, 2000, 32(4): 677-695.
68. Kaab S, Zwermann L, Barth A, et al. Selective block of sarcolemmal IKATP in human cardiomyocytes using HMR 1098 [J]. Cardiovasc Drugs Ther, 2003, 17 (5-6): 435-441.
69. Wang S.Y., Wang G.K.. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins [J]. Cell signal, 2003, 15(2): 151 -159.
70. A.D. Zechnich, J.R. Hedges, D Eiselt-Proteau, et al. Possible interactions with terfenadine or astemizole [J]. West J Med, 1994, 160(4): 321-325.
71．董晞，赵世萍，刘岩，等．甘草苷对乌头碱致心肌细胞损伤的保护作用[J]．中华中医药杂志，2009，24(2)：163-166．
72. Hilgemann D W, Nicoll D A, Philipson K D. Change movement during Na~+ translocation by native and cloned cardiac Na~+/Ca~(2+) exchanger [J]. Nature, 1991, 352(6337): 715-718.
73. Bridge J H B, Smolley J R, Spitzer K W. The relationship between charge movements associated with I_(Ca) and I_(Na-Ca) in cardiac myocytes [J]. Science, 1990, 248(4953): 376-378.
74. Amran M S, Homma N, Hashimoto K. Pharmacology of KB-R7943: a Na~+-Ca~(2+) exchange inhibitor [J]. Cardiovasc. Drug Rev., 2003, 21(4): 255-276.
75. Amran MS, Hashimoto K, Homma N. Effects of sodium-calcium exchange inhibitors, KB-R7943 and SEA0400, on aconitine-induced arrhythmias in guinea pigs in vivo, in vitro, and in computer simulation studies [J]. J. Pharmacol. Exp. Ther., 2004, 310(1): 83-89.
76. Liu Y, Zhang SW, Liang M, et al. Effects of aconitine on [Ca~(2+)] oscillation in cultured myocytes of neonatal rats [J]. J Huazhong Univ Sci Technolog, 2008, 28(5): 499-503.
77. Harberd N, Artavanis-Tsakonas S, Berridge MJ, Bourne H, Cantley L, Downward J, et al. Cell Communication. In: Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, eds. Molecular Biology of the Cell[M]. Garland Science, 4~(th) Ed. New York, NY: Taylor & Francis Group, 2002, 831-906.
78. Harberd N, Artavanis-Tsakonas S, Berridge M J, et al. Cell Communication. In: Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, eds. Molecular Biology of the Cell. Garland Science, 4~(th) Ed. New York, NY: Taylor & Francis Group, 2002, 831-906.
79. Van Rijen HVM, Eckardt D, Degen J. et al. Slow conduction and enhanced anisotropy increase the propensity for ventricular tachyarrhythmias in adult mice with induced deletion of connexin43 [J]. Circulation, 2004, 109(8): 1048-1055.
80. Zhang Y, Kakinuma Y, Ando M, et al. Acetylcholine inhibits the hypoxia-induced reduction of connexin43 protein in rat cardiomyocytes [J]. J Pharmacol Sci, 2006, 101(3): 214-222.
81. Gutstein D E, Morley G E, Tamaddon H, et al. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43 [J]. Circ. Res., 2001, 88(3): 333-339.
82. Dhein S. Pharmacology of gap junctions in the cardiovascular system [J]. Cardiovasc Res, 2004, 62(2): 287-298.
83．王云胜，林吉进．连接蛋白43磷酸化状态与心脏缝隙连接通道功能的关系[J]．国际心血管病杂志，2007，34(4)：270-273．
84. Axelsen LN, Stahlhut M, Mohammed S, et al. Identification of ischemia-regulated phosphorylation sites in connexin43: a possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123) [J]. J Mol Cell Cardiol, 2006, 40(6): 790-798.
85. Zhang S-W, Liu Y, Huang G-Z, et al. Aconitine alters connexin43 phosphorylation status and [Ca~(2+)] oscillation patterns in cultured neonatal rat ventricular myocytes [J]. Toxicol. In Vitro, 2007, 21(8): 1476-1485.
86. Beardslee MA, Lerner DL, Tadros PN, et al. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia [J]. Circ Res, 2000, 87(8): 656-662.
87. Matsushita S, Kurihara H, Watanabe M, et al. Alterations of phosphorylation state of connexin43 during hypoxia and reoxygenation are associated with cardiac function [J]. J Histochem Cytochem, 2006, 54(3): 343-353.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |