大鼠Ⅲ度房室传导阻滞模型中慢病毒介导的RNA干扰抑制心室肌KCNJ2基因表达对心室率影响的研究
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摘要
研究背景
人工电子心脏起搏器(cardiac pacemaker)作为治疗某些心律失常(主要是缓慢性心律失常)时首选的治疗仪器,是通过一种植入于人体内的电子脉冲发生器发放电脉冲,进而使与电极所接触的心肌细胞产生有节律的冲动,使心脏在此节律下有规律的激动和收缩,从而达到治疗的目的。自从1958年Rune Elmqvist成功将制造出的第一台人工心脏起搏器植入到患者体内至今,在缓慢型心律失常(如病窦综合征和重度房室传导阻滞等)治疗方面,人工起搏器已经被公认为首选的治疗方法。不过在人们的应用过程中,人工起搏器自身存在的一些缺陷及问题不断暴露出来,其中比较严重的包括：电极易于发生断裂、外来植入物可能导致感染、静脉内血栓形成而导致的栓塞、电池维持时间有限需重新进行手术更换等。除此之外,对于特殊患者特别是儿童而言,随着患儿的不断成长和发育,早先所安装的起搏器会逐渐无法满足机体的需要,因此大多需要进行重新更换,这都限制了它在某些特定患者中的应用。
近些年随着分子生物学的发展,为了克服人工起搏器所存在的以上问题,有学者提出了生物起搏器的概念并被寄予厚望。目前,构建生物起搏器的方法主要包括细胞治疗和基因治疗两大方向,但是因为细胞治疗存在的一些问题如：伦理问题、成瘤问题、免疫排斥问题以及效果欠佳等,限制了其在这一领域的应用。而通过基因治疗构建心脏生物起搏器却越来越引起相关研究者的重视,现在基因治疗主要包括以下三种方式：(1)通过过度表达p2肾上腺素受体基因来增加心房的电活动；(2)过度表达或移植人工制造的工程化起搏电流(HCN2)基因来构建起搏细胞；(3)通过基因技术抑制内向整流钾电流(Ikl),引起心肌细胞钾电流的平衡发生变化,进而使无起搏活性的心肌细胞产生自律性。
心肌是由心肌细胞构成的一种肌肉组织,广义上说,除了包括我们熟悉的具有收缩功能的工作细胞心房肌和心室肌以外,还包括无收缩功能的窦房结、结间束、房室交界部、房室束(即希斯束)和浦肯野纤维等5种特殊分化了的心肌细胞。后5种心肌细胞没有收缩功能,但是具有自律性和传导性,它们共同组成心脏的起搏和传导系统,是心脏能够进行自律性活动的结构基础；前两种心肌细胞具有收缩性,是心脏舒缩活动的功能基础。早期研究发现,成人心室肌细胞有潜在的起搏活性；另外在心肌Ikl中KCNJ2基因所编码的通道蛋白Kir2.1占80%左右,它可以使成人心室肌细胞的静息膜电位保持在负电位水平,而且它高度表达于没有自律性的心房肌和心室肌中,而在窦房结等具备自律性的细胞中表达却相对罕见或缺乏。因此有学者认为,由于受到Ikl的影响,心室肌的起搏活性才会被抑制。以上观点在后期的实验中得到了证实,其中在Silva J等的研究中发现,当对心室肌细胞的Ikl电流的抑制率达到81%以后,其潜在的起搏活性就会显现出来,出现自发的动作电位,而且其自发的搏动频率会随着Ik1电流抑制率的增加而相应的提高。而Miake等则应用基因工程构造出编码没有功能、非活性的IK1通道的Kir2.1AAA基因,通过负显性法来抑制心室肌细胞的Ikl电流。研究中他们发现在没有对豚鼠心脏正常兴奋性进行负性干预的前提下,成功的诱导出了起源于豚鼠心室肌细胞的自发起搏活动,而且经过检测此起搏活动的频率比窦房结的正常频率还要快,被认为是世界上首例成功构建的生物心脏起搏器。另外,有研究者通过动物实验还发现,如果将心肌细胞中KCNJ2基因的表达完全阻断,会导致多种心律失常的出现。Chan YC等进一步的研究则发现,在生物起搏器的基因治疗中抑制Kir2.1蛋白表达与过表达HCN4基因有协同作用,可以共同提高心肌细胞的自律性。近期,位于美国的Cedars-Sinai心脏研究所的KapoorN等通过向普通心肌细胞中注入单个Tbx18基因对其进行重新编程,将心肌细胞改造成专业化很高的“生物起搏器”的实验获得了成功,另外他们还发现改造后的心肌细胞所产生的电活动,可以规律的传导至全部心肌细胞,引起心脏出现有节奏的舒缩,证实了通过植入单个基因即可以将心肌细胞改造成专门的起搏细胞,并且通过心室的这个起搏点能够引起心脏出现规律的肌肉活动。这一事实进一步为通过提高局部心室肌细胞自律性来在体构建生物起搏器提供了依据。
RNA干扰(RNA interference, RNAi)技术是目前比较成熟的一种基因干预技术,是由双链RNA(dsRNA)诱导的同源mRNA降解的过程[14,15]。能够达到在转录水平、转录后水平和翻译水平上特异性阻断基因表达的效果。目前人们已经掌握了根据RNA干扰的原理人工合成siRNAs (small interference RNA)的技术,为通过使用能表达siRNAs的载体在体外或体内诱导哺乳动物的细胞产生特异的基因沉默提供了技术支持。
慢病毒载体(Lentiviral vector,LVs)是在人免疫缺陷病毒1型(HIV-1)基础上改造而成的,能利用逆转录酶和整合酶,将自身的RNA转变为DNA整合到宿主细胞的染色体中,从而使目的基因在宿主细胞中长期而稳定的表达。因为其具有宿主范围广,所能容纳的外源性基因片段大,安全性高等优点,目前在基因治疗中的应用越来越广泛。
前期本课题组已经获得了干扰KCNJ2基因mRNA的特异位点(+361～+379碱基),并且通过应用设计出的特异干扰序列,在细胞水平证实了将RNA干扰基因导入心室肌细胞,使其产生干扰效应,特异性得使KCNJ2基因沉默,抑制内向整流钾电流(Ik1),进而诱导处于静息状态的心室肌细胞产生生物起搏活性的可行性。
在本课题中,我们将在动物体内通过干扰KCNJ2基因来抑制模型大鼠心肌的内向整流钾电流(Ik1),探讨是否可以提高模型大鼠的心室率。在实验中我们通过建立大鼠Ⅲ度房室传导阻滞的模型,消除窦性起搏对心室肌细胞自律性的掩盖；进而应用慢病毒载体介导的RNAi技术,通过干扰KCNJ2基因从而抑制Kir2.1蛋白,达到抑制内向整流钾电流(Ik1)通道的目的,观察模型大鼠心室率的变化情况来探讨以上假设的可行性。希望可以为生物起搏器的体内研究提供一些有价值的试验依据。
第一部分携带有特异性干扰片段的慢病毒载体的构建
研究目的
制备含有针对大鼠心肌细胞KCNJ2基因nRNA的特异性shRNA的慢病毒表达载体。
研究方法
1、将前期验证的对KCNJ2基因mRNA上沉默效果最明显的干扰片断以MluI,ClaI为插入位点,插入到由H1启动子调控以及有GFP表达的慢病毒载体中；并通过MluI,ClaI双酶切技术进行鉴定。
2、在脂质体的介导下将混合的慢病毒载体包装质粒和包含KCNJ2基因干扰片段的重组慢病毒载体共转染293T细胞,包装成病毒,72小时后收集上清,应用逐孔稀释滴度测定法测定病毒滴度。研究结果
通过双酶切电泳证实所设计的KCNJ2基因shRNA正确插入到了慢病毒载体中,DNA测序证实插入的序列正确；293T细胞成功包装重组慢病毒载体；收集的细胞培养上清液中,病毒的滴度为1.07×109TU/ml。结论
制备的慢病毒载体构建成功,同时获得的病毒滴度较高,完全满足后续在体实验的要求。
第二部分在大鼠III度房室传导阻滞模型中抑制心室肌KCNJ2基因的表达对心室率的影响
研究目的
1、建立稳定的大鼠III度房室传导阻滞的模型。
2、确定慢病毒载体转染大鼠的最佳滴度。
3、明确慢病毒载体转染大鼠III度房室传导阻滞模型后心室率的变化情况。
4、明确慢病毒载体转染心肌细胞后KCNJ2基因mRNA和Kir2.1蛋白表达的变化及其于大鼠心室率的关系。研究方法
1、采用定点注射70%乙醇的方法破坏大鼠的房室结,使大鼠发生III度房室传导阻滞。注射乙醇约25u1,观察心电图变化,出现III度房室传导阻滞的大鼠观察30分钟,待稳定后关胸,72小时后检测确定建模是否成功。
2、取正常大鼠给予不同滴度的空白慢病毒载体30ul进行心肌注射,分别取注射后第4天、7天、10天处死取材,迅速制作冰冻切片,在免疫荧光显微镜下进行检测,根据各不同时段切片中的绿色荧光蛋白的亮度,确定转染效率与病毒滴度的关系,用转染效率最高的滴度进行模型大鼠的注射。
3、将建立的模型大鼠分为对照组、空载体组、病毒干预组三组；其中对照组只进行转染的手术操作但不进行相应的病毒转染；另两组则分别转染慢病毒空载体及携带有相应shRNA的慢病毒载体。转染后分别于不同时间点通过心电图检测各组大鼠心室率的变化情况。
4、分别对各组所取得的心肌组织进行相应处理,通过实时荧光定量RT-PCR检测KCNJ2基因的表达情况；应用Western-blot及免疫组化检测心肌组织中kir2.1蛋白的表达情况。研究结果
1、建立了持久、稳定的III度房室传导阻滞大鼠模型,满足本实验的要求。
2、慢病毒的最佳转染滴度为109TU/ml,转染7天后转染效率变稳定。
3、本研究发现病毒干预组中有54.5%的大鼠心室率由156±6次／分钟提高到218±10次／分钟,差异性显著(p<0.01)；进一步实验证实心室率的提高与KCNJ2基因、Kir2.1蛋白的表达呈负相关；是由慢病毒转染的基因沉默引起的,且在转染后14天,当KCNJ2基因与Kir2.1蛋白抑制率分别为77%、55%左右时,大鼠的心室率达最快。因此证实了通过抑制心肌的KCNJ2基因及Kir2.1蛋白的表达,进而抑制内向整流钾电流(Ikl),能够引起III度房室传导阻滞模型大鼠心室肌细胞自律性的提高。结论
在动物体内通过抑制KCNJ2基因来抑制模型大鼠心肌细胞的内向整流钾电流(Ikl)来提高大鼠的心室率是可行的。
BACKGROUND
Artificial electronic cardiac pacemaker is an electronic instrument implanting into the inside of the body.The electrical pulse produced by pulse generator which stimulating the adjacent myocardial cells, it make the heart exciting and contraction, so as to achieve treatment for some arrhythmia (mainly bradycardiac arrhythmia). Since the first cardiac pacemaker implantation in the human body in1958, it has gradually become the preferred treatment of slow arrhythmia such as sick sinus syndrome and severe atrioventricular block. With the pacemaker continuous improvement, its clinical indications is extended continuously, which gradually began to apply to the tachyarrhythmia and non electrical diseases, such as preventing paroxysmal atrial tachyarrhythmia, carotid sinus syncope, refractory congestive heart failure by biventricular pacing, etc. But in the process of its application some defects and problems have become obvious, such as limited battery life, infections, reoperations and venous thromoembolus,etc. Furthermore, they are not suitable for patients that apt to infection or being too young. With the progress of molecular biology in recent years, people try to take advantage of life science and technology research to develop a biological pacemaker to replace electronic pacemaker in order to overcome the above problems. The new technigue could repair or replace the native cardic pacemaker and the damaged conduction tissue to restore the heart pacemaker and conduction function.
At present, there are two major strategies in developing biological pacemaker:gene therapy and cell therapy. Cell therapy is definited as the method of applying stem cells (embryonic stem cells and mesenchymal stem cells) and sinus node cells. But there are lots of unsolved problems such as:immunne rejection, oriented differation, tumorigenicity and ethinic issue, etc. Developing biological pacemaker by gene therapy is rooted on three strategies:(1) Over-expressing the neurohormone receptors to increase the atria electric activity.(2)Over-expressing the HCN2in diastolic phase.(3) Suppressing the inward-rectifier potassium current(Ikl) to break the balance of the potassium currenct inside the ventricular cells, which then can obtain the capability of automatic rhythmicity.
Myocardium is a muscle tissue that is composed of myocardial cells.The generalized myocardial cells not only included atrial and ventricular muscle but also the S-A node,internodal bundle, atrioventricular bundle, atrioventricular junction area and Purkinje fibers. The previously study demonstrates that adult myocardial cells possess the latent of pacing ability, which it is inhibited by IK1. With the aid of powerful inward rectifier properties holding the rest potential at negative level, IK1then is able to inhibit the myocyte spontaneous depolarization. IK1potassium is coded by gene KCNJ2, abundant in atrial and ventricular myocytes while sinus node cell is lack of this kind of channel. It is then assumed that ventricular myocyte can be changed to pacemaker cell if the IK1is inhibited. Silva J and Rudy Y found that after suppression Ikl by81%, the ventricular myocytes will generate a spontaneous action; and the more on IK1is inhibated, the higher the pacing rates of myocytes is. In2002, Miake reported a biological pacemaker created by dominant-negative therapy. They got spontaneous ventricular rhythm which was more rapid than that caused by the native sinus pacemaker. The beating rates of myocytes can also respond positively to β receptors agonist, creating rudiment for the future improvment of biological pacemaking. However, in the phenotype of completely lack of IK1, the mice bears Anderson's syndroms, such as QT-prolongatioin, periodic paralysis, skeletal and craniofacial abnormalities.
RNA interference (RNAi) is a phenomenon of gene silencing at the lever of post transcription resulted from the degradation of mRNA reduced by double strands RNA.RNAi was firstly founded in Caenorhabditis elegans. Recently,with the deepening of the research on RNAi, people have been able to induce specific gene silencing of mammalian cells by way of artificial siRNA (small interference RNA) synthesized according to the principle of RNAi in vitro or expressed through vector in vivo.
Lentiviral vector was transformated on the basis of HIV-1. Lentiviral vector could mediate exogenous gene expression in host cell sustained.It has many advantages, such as wide host range, more stable, accommodate bigger exogenous gene fragment, high security and so on. Thus, the lentiviral vector has become the most used vector in gene therapy.
In our previous studies, we has screened out the most significant suppressing sites on KCNJ2gene mRNA,namely+361-+379bp;and found it is feasible to increasethe heart rate by using RNAi to knock down the KCNJ2targeting gene in vitro.
In this study we want to use RNA interference to knock down the KCNJ2targeting gene in vivo.We observed the effect of siRNAs transfection to inhibit the KCNJ2gene expression on ventricular rate in the Ⅲ°AVB rat model, so as to provide a new effective idea and approach for the study of biological pacemaker.
Part1. Construction of lentiviral vector with the shRNA effect to KCNJ2
Objective
To construct specific lentiviral vector with the shRNA effect to rat cardiomyocytes gene KCNJ2mRNA.
Method
According to our previous research, the double-stranded DNA oligo which can cause most RNAi effect to KCNJ2was made. The small hairpin RNA(shRNA) sequences were annealed and linked with linearized into the lentiviral vector. Moreover, the recombinant lentivirus was harvested from293T cells when it cotransfected with lentiviral packing materials into them after72h. The virus particles were collected. Virus titer was determined by hole dilution method.
Result
The shRNA sequences were successfully inserted into lentiviral vector by double restriction digestion, and the sequences were identified by DNA sequencing. The shRNA of KCNJ2gene of the recombinant lentiviral vector was successfully packed into293T cells. The recombinant lentivirus was harvested from293T cells, and the titer of the virus of1.07x109TU/ml.
conclusion
The lentiviral vector with the shRNA effect to rat cardiomyocytes gene KCNJ2mRNA was successfully constructed.
Part2. Experimental study on inhibition of ventricular Ikl by RNA interference targeting the KCNJ2gene in three degree atrioventricular block rat
Objective
1.To establish a stable rat three degree atrioventricular block(Ⅲ°AVB) model;
2.To confirm the best virus titer to lentiviral vector infection in rat;
3.To observe the variation of the heart rate(HR)after the lentiviral vector infection;
4.To observe the alteration of the expression of KCNJ2gene and the Kir2.1protein after the lentiviral vector infection.
Method
1.A50ul needle was used to inject the solutions into the myocardium toward the nodal tissue,When the insertion of the needle resulted in momentary complete AV block,25ul of70%ethanol were injected. Hearts were reinjected with ethanol if the heart block resolved after30min. After72hours, the Ⅲ°AVB was defined as stable;
2.The rats were infected by lentiviral vector of different virus titer; we drawn material at different time and made frozen sections. the infection rate was detected by immunofluoresc-ence microscopy, then confirmed the best virus titer to lentiviral vector infection in rat;
3.The Ⅲ°AVB rat models were divided into three groups (1) Interference group: infected lentiviral vector with shRNA;(2)Negative control group:infected negative lentiviral vector;(3) Controll group:no treatment.The HR was analyzed with electrical cardiogram;
4.The levels of target genes mRNA and protein were analyzed with the real time quantitative (RT-PCR), Western blot, Immunity histochemistry.
Result
1.the Ⅲ°AVB rat model was stable;
2.The best virus titer to lentiviral vector infection in rat was1×10TU/ml;
3.The HR of rat was increase after the lentiviral vector infection;The expression of KCNJ2gene and Kir2.1protein were suppressed after the lentiviral vector infection.
Conclusion
It is feasible to increase the HR by using RNAi to knock down the KCNJ2targeting gene in vivo.
人工电子心脏起搏器(cardiac pacemaker)作为治疗某些心律失常(主要是缓慢性心律失常)时首选的治疗仪器,是通过一种植入于人体内的电子脉冲发生器发放电脉冲,进而使与电极所接触的心肌细胞产生有节律的冲动,使心脏在此节律下有规律的激动和收缩,从而达到治疗的目的。自从1958年Rune Elmqvist成功将制造出的第一台人工心脏起搏器植入到患者体内至今,在缓慢型心律失常(如病窦综合征和重度房室传导阻滞等)治疗方面,人工起搏器已经被公认为首选的治疗方法。不过在人们的应用过程中,人工起搏器自身存在的一些缺陷及问题不断暴露出来,其中比较严重的包括：电极易于发生断裂、外来植入物可能导致感染、静脉内血栓形成而导致的栓塞、电池维持时间有限需重新进行手术更换等。除此之外,对于特殊患者特别是儿童而言,随着患儿的不断成长和发育,早先所安装的起搏器会逐渐无法满足机体的需要,因此大多需要进行重新更换,这都限制了它在某些特定患者中的应用。
近些年随着分子生物学的发展,为了克服人工起搏器所存在的以上问题,有学者提出了生物起搏器的概念并被寄予厚望。目前,构建生物起搏器的方法主要包括细胞治疗和基因治疗两大方向,但是因为细胞治疗存在的一些问题如：伦理问题、成瘤问题、免疫排斥问题以及效果欠佳等,限制了其在这一领域的应用。而通过基因治疗构建心脏生物起搏器却越来越引起相关研究者的重视,现在基因治疗主要包括以下三种方式：(1)通过过度表达p2肾上腺素受体基因来增加心房的电活动；(2)过度表达或移植人工制造的工程化起搏电流(HCN2)基因来构建起搏细胞；(3)通过基因技术抑制内向整流钾电流(Ikl),引起心肌细胞钾电流的平衡发生变化,进而使无起搏活性的心肌细胞产生自律性。
心肌是由心肌细胞构成的一种肌肉组织,广义上说,除了包括我们熟悉的具有收缩功能的工作细胞心房肌和心室肌以外,还包括无收缩功能的窦房结、结间束、房室交界部、房室束(即希斯束)和浦肯野纤维等5种特殊分化了的心肌细胞。后5种心肌细胞没有收缩功能,但是具有自律性和传导性,它们共同组成心脏的起搏和传导系统,是心脏能够进行自律性活动的结构基础；前两种心肌细胞具有收缩性,是心脏舒缩活动的功能基础。早期研究发现,成人心室肌细胞有潜在的起搏活性；另外在心肌Ikl中KCNJ2基因所编码的通道蛋白Kir2.1占80%左右,它可以使成人心室肌细胞的静息膜电位保持在负电位水平,而且它高度表达于没有自律性的心房肌和心室肌中,而在窦房结等具备自律性的细胞中表达却相对罕见或缺乏。因此有学者认为,由于受到Ikl的影响,心室肌的起搏活性才会被抑制。以上观点在后期的实验中得到了证实,其中在Silva J等的研究中发现,当对心室肌细胞的Ikl电流的抑制率达到81%以后,其潜在的起搏活性就会显现出来,出现自发的动作电位,而且其自发的搏动频率会随着Ik1电流抑制率的增加而相应的提高。而Miake等则应用基因工程构造出编码没有功能、非活性的IK1通道的Kir2.1AAA基因,通过负显性法来抑制心室肌细胞的Ikl电流。研究中他们发现在没有对豚鼠心脏正常兴奋性进行负性干预的前提下,成功的诱导出了起源于豚鼠心室肌细胞的自发起搏活动,而且经过检测此起搏活动的频率比窦房结的正常频率还要快,被认为是世界上首例成功构建的生物心脏起搏器。另外,有研究者通过动物实验还发现,如果将心肌细胞中KCNJ2基因的表达完全阻断,会导致多种心律失常的出现。Chan YC等进一步的研究则发现,在生物起搏器的基因治疗中抑制Kir2.1蛋白表达与过表达HCN4基因有协同作用,可以共同提高心肌细胞的自律性。近期,位于美国的Cedars-Sinai心脏研究所的KapoorN等通过向普通心肌细胞中注入单个Tbx18基因对其进行重新编程,将心肌细胞改造成专业化很高的“生物起搏器”的实验获得了成功,另外他们还发现改造后的心肌细胞所产生的电活动,可以规律的传导至全部心肌细胞,引起心脏出现有节奏的舒缩,证实了通过植入单个基因即可以将心肌细胞改造成专门的起搏细胞,并且通过心室的这个起搏点能够引起心脏出现规律的肌肉活动。这一事实进一步为通过提高局部心室肌细胞自律性来在体构建生物起搏器提供了依据。
RNA干扰(RNA interference, RNAi)技术是目前比较成熟的一种基因干预技术,是由双链RNA(dsRNA)诱导的同源mRNA降解的过程[14,15]。能够达到在转录水平、转录后水平和翻译水平上特异性阻断基因表达的效果。目前人们已经掌握了根据RNA干扰的原理人工合成siRNAs (small interference RNA)的技术,为通过使用能表达siRNAs的载体在体外或体内诱导哺乳动物的细胞产生特异的基因沉默提供了技术支持。
慢病毒载体(Lentiviral vector,LVs)是在人免疫缺陷病毒1型(HIV-1)基础上改造而成的,能利用逆转录酶和整合酶,将自身的RNA转变为DNA整合到宿主细胞的染色体中,从而使目的基因在宿主细胞中长期而稳定的表达。因为其具有宿主范围广,所能容纳的外源性基因片段大,安全性高等优点,目前在基因治疗中的应用越来越广泛。
前期本课题组已经获得了干扰KCNJ2基因mRNA的特异位点(+361～+379碱基),并且通过应用设计出的特异干扰序列,在细胞水平证实了将RNA干扰基因导入心室肌细胞,使其产生干扰效应,特异性得使KCNJ2基因沉默,抑制内向整流钾电流(Ik1),进而诱导处于静息状态的心室肌细胞产生生物起搏活性的可行性。
在本课题中,我们将在动物体内通过干扰KCNJ2基因来抑制模型大鼠心肌的内向整流钾电流(Ik1),探讨是否可以提高模型大鼠的心室率。在实验中我们通过建立大鼠Ⅲ度房室传导阻滞的模型,消除窦性起搏对心室肌细胞自律性的掩盖；进而应用慢病毒载体介导的RNAi技术,通过干扰KCNJ2基因从而抑制Kir2.1蛋白,达到抑制内向整流钾电流(Ik1)通道的目的,观察模型大鼠心室率的变化情况来探讨以上假设的可行性。希望可以为生物起搏器的体内研究提供一些有价值的试验依据。
第一部分携带有特异性干扰片段的慢病毒载体的构建
研究目的
制备含有针对大鼠心肌细胞KCNJ2基因nRNA的特异性shRNA的慢病毒表达载体。
研究方法
1、将前期验证的对KCNJ2基因mRNA上沉默效果最明显的干扰片断以MluI,ClaI为插入位点,插入到由H1启动子调控以及有GFP表达的慢病毒载体中；并通过MluI,ClaI双酶切技术进行鉴定。
2、在脂质体的介导下将混合的慢病毒载体包装质粒和包含KCNJ2基因干扰片段的重组慢病毒载体共转染293T细胞,包装成病毒,72小时后收集上清,应用逐孔稀释滴度测定法测定病毒滴度。研究结果
通过双酶切电泳证实所设计的KCNJ2基因shRNA正确插入到了慢病毒载体中,DNA测序证实插入的序列正确；293T细胞成功包装重组慢病毒载体；收集的细胞培养上清液中,病毒的滴度为1.07×109TU/ml。结论
制备的慢病毒载体构建成功,同时获得的病毒滴度较高,完全满足后续在体实验的要求。
第二部分在大鼠III度房室传导阻滞模型中抑制心室肌KCNJ2基因的表达对心室率的影响
研究目的
1、建立稳定的大鼠III度房室传导阻滞的模型。
2、确定慢病毒载体转染大鼠的最佳滴度。
3、明确慢病毒载体转染大鼠III度房室传导阻滞模型后心室率的变化情况。
4、明确慢病毒载体转染心肌细胞后KCNJ2基因mRNA和Kir2.1蛋白表达的变化及其于大鼠心室率的关系。研究方法
1、采用定点注射70%乙醇的方法破坏大鼠的房室结,使大鼠发生III度房室传导阻滞。注射乙醇约25u1,观察心电图变化,出现III度房室传导阻滞的大鼠观察30分钟,待稳定后关胸,72小时后检测确定建模是否成功。
2、取正常大鼠给予不同滴度的空白慢病毒载体30ul进行心肌注射,分别取注射后第4天、7天、10天处死取材,迅速制作冰冻切片,在免疫荧光显微镜下进行检测,根据各不同时段切片中的绿色荧光蛋白的亮度,确定转染效率与病毒滴度的关系,用转染效率最高的滴度进行模型大鼠的注射。
3、将建立的模型大鼠分为对照组、空载体组、病毒干预组三组；其中对照组只进行转染的手术操作但不进行相应的病毒转染；另两组则分别转染慢病毒空载体及携带有相应shRNA的慢病毒载体。转染后分别于不同时间点通过心电图检测各组大鼠心室率的变化情况。
4、分别对各组所取得的心肌组织进行相应处理,通过实时荧光定量RT-PCR检测KCNJ2基因的表达情况；应用Western-blot及免疫组化检测心肌组织中kir2.1蛋白的表达情况。研究结果
1、建立了持久、稳定的III度房室传导阻滞大鼠模型,满足本实验的要求。
2、慢病毒的最佳转染滴度为109TU/ml,转染7天后转染效率变稳定。
3、本研究发现病毒干预组中有54.5%的大鼠心室率由156±6次／分钟提高到218±10次／分钟,差异性显著(p<0.01)；进一步实验证实心室率的提高与KCNJ2基因、Kir2.1蛋白的表达呈负相关；是由慢病毒转染的基因沉默引起的,且在转染后14天,当KCNJ2基因与Kir2.1蛋白抑制率分别为77%、55%左右时,大鼠的心室率达最快。因此证实了通过抑制心肌的KCNJ2基因及Kir2.1蛋白的表达,进而抑制内向整流钾电流(Ikl),能够引起III度房室传导阻滞模型大鼠心室肌细胞自律性的提高。结论
在动物体内通过抑制KCNJ2基因来抑制模型大鼠心肌细胞的内向整流钾电流(Ikl)来提高大鼠的心室率是可行的。
BACKGROUND
Artificial electronic cardiac pacemaker is an electronic instrument implanting into the inside of the body.The electrical pulse produced by pulse generator which stimulating the adjacent myocardial cells, it make the heart exciting and contraction, so as to achieve treatment for some arrhythmia (mainly bradycardiac arrhythmia). Since the first cardiac pacemaker implantation in the human body in1958, it has gradually become the preferred treatment of slow arrhythmia such as sick sinus syndrome and severe atrioventricular block. With the pacemaker continuous improvement, its clinical indications is extended continuously, which gradually began to apply to the tachyarrhythmia and non electrical diseases, such as preventing paroxysmal atrial tachyarrhythmia, carotid sinus syncope, refractory congestive heart failure by biventricular pacing, etc. But in the process of its application some defects and problems have become obvious, such as limited battery life, infections, reoperations and venous thromoembolus,etc. Furthermore, they are not suitable for patients that apt to infection or being too young. With the progress of molecular biology in recent years, people try to take advantage of life science and technology research to develop a biological pacemaker to replace electronic pacemaker in order to overcome the above problems. The new technigue could repair or replace the native cardic pacemaker and the damaged conduction tissue to restore the heart pacemaker and conduction function.
At present, there are two major strategies in developing biological pacemaker:gene therapy and cell therapy. Cell therapy is definited as the method of applying stem cells (embryonic stem cells and mesenchymal stem cells) and sinus node cells. But there are lots of unsolved problems such as:immunne rejection, oriented differation, tumorigenicity and ethinic issue, etc. Developing biological pacemaker by gene therapy is rooted on three strategies:(1) Over-expressing the neurohormone receptors to increase the atria electric activity.(2)Over-expressing the HCN2in diastolic phase.(3) Suppressing the inward-rectifier potassium current(Ikl) to break the balance of the potassium currenct inside the ventricular cells, which then can obtain the capability of automatic rhythmicity.
Myocardium is a muscle tissue that is composed of myocardial cells.The generalized myocardial cells not only included atrial and ventricular muscle but also the S-A node,internodal bundle, atrioventricular bundle, atrioventricular junction area and Purkinje fibers. The previously study demonstrates that adult myocardial cells possess the latent of pacing ability, which it is inhibited by IK1. With the aid of powerful inward rectifier properties holding the rest potential at negative level, IK1then is able to inhibit the myocyte spontaneous depolarization. IK1potassium is coded by gene KCNJ2, abundant in atrial and ventricular myocytes while sinus node cell is lack of this kind of channel. It is then assumed that ventricular myocyte can be changed to pacemaker cell if the IK1is inhibited. Silva J and Rudy Y found that after suppression Ikl by81%, the ventricular myocytes will generate a spontaneous action; and the more on IK1is inhibated, the higher the pacing rates of myocytes is. In2002, Miake reported a biological pacemaker created by dominant-negative therapy. They got spontaneous ventricular rhythm which was more rapid than that caused by the native sinus pacemaker. The beating rates of myocytes can also respond positively to β receptors agonist, creating rudiment for the future improvment of biological pacemaking. However, in the phenotype of completely lack of IK1, the mice bears Anderson's syndroms, such as QT-prolongatioin, periodic paralysis, skeletal and craniofacial abnormalities.
RNA interference (RNAi) is a phenomenon of gene silencing at the lever of post transcription resulted from the degradation of mRNA reduced by double strands RNA.RNAi was firstly founded in Caenorhabditis elegans. Recently,with the deepening of the research on RNAi, people have been able to induce specific gene silencing of mammalian cells by way of artificial siRNA (small interference RNA) synthesized according to the principle of RNAi in vitro or expressed through vector in vivo.
Lentiviral vector was transformated on the basis of HIV-1. Lentiviral vector could mediate exogenous gene expression in host cell sustained.It has many advantages, such as wide host range, more stable, accommodate bigger exogenous gene fragment, high security and so on. Thus, the lentiviral vector has become the most used vector in gene therapy.
In our previous studies, we has screened out the most significant suppressing sites on KCNJ2gene mRNA,namely+361-+379bp;and found it is feasible to increasethe heart rate by using RNAi to knock down the KCNJ2targeting gene in vitro.
In this study we want to use RNA interference to knock down the KCNJ2targeting gene in vivo.We observed the effect of siRNAs transfection to inhibit the KCNJ2gene expression on ventricular rate in the Ⅲ°AVB rat model, so as to provide a new effective idea and approach for the study of biological pacemaker.
Part1. Construction of lentiviral vector with the shRNA effect to KCNJ2
Objective
To construct specific lentiviral vector with the shRNA effect to rat cardiomyocytes gene KCNJ2mRNA.
Method
According to our previous research, the double-stranded DNA oligo which can cause most RNAi effect to KCNJ2was made. The small hairpin RNA(shRNA) sequences were annealed and linked with linearized into the lentiviral vector. Moreover, the recombinant lentivirus was harvested from293T cells when it cotransfected with lentiviral packing materials into them after72h. The virus particles were collected. Virus titer was determined by hole dilution method.
Result
The shRNA sequences were successfully inserted into lentiviral vector by double restriction digestion, and the sequences were identified by DNA sequencing. The shRNA of KCNJ2gene of the recombinant lentiviral vector was successfully packed into293T cells. The recombinant lentivirus was harvested from293T cells, and the titer of the virus of1.07x109TU/ml.
conclusion
The lentiviral vector with the shRNA effect to rat cardiomyocytes gene KCNJ2mRNA was successfully constructed.
Part2. Experimental study on inhibition of ventricular Ikl by RNA interference targeting the KCNJ2gene in three degree atrioventricular block rat
Objective
1.To establish a stable rat three degree atrioventricular block(Ⅲ°AVB) model;
2.To confirm the best virus titer to lentiviral vector infection in rat;
3.To observe the variation of the heart rate(HR)after the lentiviral vector infection;
4.To observe the alteration of the expression of KCNJ2gene and the Kir2.1protein after the lentiviral vector infection.
Method
1.A50ul needle was used to inject the solutions into the myocardium toward the nodal tissue,When the insertion of the needle resulted in momentary complete AV block,25ul of70%ethanol were injected. Hearts were reinjected with ethanol if the heart block resolved after30min. After72hours, the Ⅲ°AVB was defined as stable;
2.The rats were infected by lentiviral vector of different virus titer; we drawn material at different time and made frozen sections. the infection rate was detected by immunofluoresc-ence microscopy, then confirmed the best virus titer to lentiviral vector infection in rat;
3.The Ⅲ°AVB rat models were divided into three groups (1) Interference group: infected lentiviral vector with shRNA;(2)Negative control group:infected negative lentiviral vector;(3) Controll group:no treatment.The HR was analyzed with electrical cardiogram;
4.The levels of target genes mRNA and protein were analyzed with the real time quantitative (RT-PCR), Western blot, Immunity histochemistry.
Result
1.the Ⅲ°AVB rat model was stable;
2.The best virus titer to lentiviral vector infection in rat was1×10TU/ml;
3.The HR of rat was increase after the lentiviral vector infection;The expression of KCNJ2gene and Kir2.1protein were suppressed after the lentiviral vector infection.
Conclusion
It is feasible to increase the HR by using RNAi to knock down the KCNJ2targeting gene in vivo.
引文
1.顾江英,朱平。基因治疗疾病的憧憬与挫折[J].自然杂志,2004；26(2)；103-107。
2. Aiuti A, Roncarolo MG. Ten years of gene therapy for primary immune deficiencies.Hematology Am Soc Hematol Educ Program [J].2009:682-9.
3. Mukherjee S, Thrasher AJ.Gene therapy for PIDs:progress, pitfalls and prospects [J].Gene.2013 Aug 10;525(2):174-81
4. Howe SJ, Mansour MR, Schwarzwaelder K,et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients [J]. J Clin Invest.2008 Sep;118(9):3143-50
5. Weber E, Anderson WF, Kasahara N. Recent advances in retrovirus vector-mediated gene therapy:teaching an old vector new tricks [J]. Curr Opin Mol Ther.2001 Oct;3(5):439-53.
6.杨力明,曲章义,侯咏等。人3型腺病毒六邻体基因表达质粒的构建及表达[J].哈尔滨医科大学学报,2004,38(3)：218-220.
7. Liu L,Pedersen E F. Partial Abstracts of the 3rd Annual Meeting of the China RNA Society & 1st International RNA Workshop in China:SESSION 1: mRNA PROCESSING-Biochemical Identification of Nuclear Proteins p30 Specifically Interacting with Upstream Repressi [J]. Acta Biochimica et Biophysica Sinica,2004,36(2):147.
8. Zhang QY, Jin YZ, Zhang HB, Xie SS.Construction and biological activities of CTLA4Ig adenovirus vectors [J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2003 Jan;19(1):45-8.
9.李方成,李军亮,吴中华等.大鼠GLUT3基因重组腺病毒载体的构建及鉴定[J].中华神经医学杂志,2006,5(11)：1086-1089.
10. Scibetta A G, Copier J, Barrett A, et al. Gene expression changes induced by a recombinant E1/E3 adenovirus type 5 vector in human mammary epithelial cells [J]. Intervirology England,2005,48 (6):350-361.
11. Schowalter D B, Matern D, Vockley J, et al. In vitro correction of medium chain acyl Co A dehydrogenase deficiency with a recombinant adeno viral vector [J] Mol Genet Metab,2005,85(2):88-95.
12.邱峰,陈世知,陈虹.新型腺病毒载体的制备及其特性研究[J]。重庆医科大学学报,2006,31(3)：330-333.
13. Baltimore D, Verma I M, Drost S, et al. Temperature-sensitive DNA polymerase from Rous sarcoma virus mutants [J]. Cancer,1974,34:1-7
14. Esposito D, Craigie R. HIV integrase structure and function [J]. Adv Virus Res, 1999,52:319-333
15. Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector [J]. Science,1996,272: 263-267
16. Mautino MR. Lentiviral vectors for gene therapy of HIV-1 infection [J]. Curr Gene Ther,2002,2(1):23-43
17. Punzon I, Criado L M, Serrano A, et al. Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background [J]. Blood,2004, 103:580-582
18. Jakobsen M, Stenderup K, Rosada C, et al. Amelioration of psoriasis by anti-TNF-alpha RNAi in the xenograft transplantation model [J]. Mol Ther,2009,17: 1743-1753
19.赵世巧。基因治疗的病毒载体研究进展。国外医学临床生物化学与检验学分册2005年10月第26卷第10期。
20. Clare E, Thomas, et al. Progress and problems with the use of viral vectors for gene therapy. Nature review genetics.2003,4:346-358.
21. David Peel. Virus Vectors & Gene Therapy:Problems, Promises & Prospects [J]. MB Ch B Special Study Module Project Report
22. PL Sinn, SL Sauter, PB McCray Jr. Gene Therapy Progress and Prospects: Development of improved lentiviral and retro viral vectors-design, biosafety, and production [J]. Gene Therapy(2005)12,1089-1098.
23. Dann, Christina Tenenhaus. New technology for an old favorite:lentiviral transgenesis and RNAi in rats [J]. Transgenic Research. October 2007,16(5): 571-580.
24. Jakobsson, et al. Efficient transduction of neurons using Ross River glycoprotein pseudotyped lentiviral vectors [J]. Gene Therapy.2006,13(12): 966-973c
25. Logan AC, Lutzko C, Kohn DB. Advances in lentiviral vector design for gene—modification of hematopoietic stem cells [J]. Current Opinion in Biotechnology 2002,13:429-436.
26.周芬,程时远。基因治疗中的非病毒载体[J]。化学通报,2005年,第68卷。
27. Kafri T, Blomer U, Peterson DA, Gage FH, Verma IM. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors [J]. Nat Genet, 1997,17(3):314-317.
28. Kafri T, van Praag H, Gage FH, Verma IM. Lentiviral vectors:regulated gene expression[J]. Moi Ther,2000,1(6):516-521.
29. Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long term expression of the transgene in adult rat brains injected with a lentiviral vector [J]. Proc Natl Acad Sci USA,1996,93(21): 11382-11388.
30. Matrai J, Chuah MK, VandenDriessche T. Recent advances in lentiviral vector development and applications [J]. Mol Ther.2010 Mar;18(3):477-90.
31. Blesch A. Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer [J]. Methods,2004,33(2):164-172.
32. Page K A, Landau N R, Littman D R. Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity [J]. J Virol,1990, 64:5270-5276.
33. Liu B, Paton JF, Kasparov S.Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use in vitro and in vivo.BMC Biotechnol.2008 May 16;8-49.
34. Emery DW.The use of chromatin insulators to improve the expression and safety of integrating gene transfer vectors [J].Hum Gene Ther. 2011 Jun;22(6):761-74.
35. Dropulic B.Lentiviral vectors:their molecular design, safety, and use in laboratory and preclinical research [J].Hum Gene Ther.2011 Jun;22(6):649-57.
36. Delenda C, Gaillard C. Real-time quantitative PGR for the design of lentiviral vector analytical assays [J]. Gene therapy 2005;12 Suppl 1:S36-50.
37. Geraerts M, Willems S, Baekelandt V et al. Comparison of lentiviral vector titration methods [J]. BMC biotechnology 2006; 6:34.
38. Chan C S, Weeber E J, Kurup S, et al Integrin requirement for hippocampal synaptic plasticity and spatial memory [J]. J Neurosci,2003,23(18):7107-7116
1. Marban E, Cho HC. Creation of a biological pacemaker by gene-or cell-based approaches [J]. Med Biol Eng Comput.2007; 45:133-144.
2. Hauser RG, Hayes DL, Kallinen LM, et al.Clinical experience with pacemaker pulse generators and transvenous leads:an 8-year prospective multicenter study [J]. Heart Rhythm 2007;4:154-160.
3. Senaratne J, Irwin ME, Senaratne MP. Pacemaker longevity:are we getting what we are promised [J]? Pacing Clin Electrophysiol 2006; 29:1044-1054.
4. Kay GN, Brinker JA, Kawanishi DT, et al. Risks of spontaneous injury and extraction of an active fixation pacemaker lead:report of the Accufix MulticenterClinical Study and Worldwide Registry [J]. Circulation 1999; 100: 2344-2352.
5. Burney K1, Burchard F, Papouchado M, et al.Cardiac pacing systems and implantable cardiac defibrillators (ICDs):a radiological perspective of equipment, anatomy and complications [J]. Clin Radiol.2004 Aug;59(8):699-708.
6. Friedman RA, Fenrich AL, Kertesz NJ. Congenital complete atrioventricular block [J].Pacing Clin Electrophysiol 2001;24:1681-1688.
7. Greene D, Kang S, Kosenko A, et al.Adrenergic regulation of HCN4 channel requires protein association with β2-adrenergic receptor [J] J Biol Chem.2012 Jul 6;287(28):23690-7.
8. Edelberg JM, Huang DT, Josephson ME, et al.Molecular enhancement of porcine cardiac chronotropy [J]. Heart.2001; 86:559-562.
9. Boink GJ, Duan L, Nearing BD, et al.HCN2/SkMl gene transfer into canine left bundle branch induces stable, autonomically responsive biological pacing at physiological heart rates [J] J Am Coll Cardiol.2013 Mar 19;61(11):1192-201.
10. Plotnikov AN, Sosunov EA, Qu J,et al.Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates [J]. Circulation 2004; 109:506-512.
11. Qu J, Barbuti A, Protas L, et al.HCN2 overexpression in newborn and adult ventricular myocytes:Distinct effects on gating and excitability [J].Circ Res. 2001;89:E8-E14.
12. Miake J, Marban E, Nuss HB. Biological pacemaker created by gene transfer [J]. Nature 2002; 419:132-133.
13. Miake J, Marban E, Nuss HB. Functional role of inward rectifier current in heart probed by Kir2.1 overexpression and dominant-negative suppression [J]. J Clin Invest.2003;111:1529-1536.
14. Paddison PJ, Caudy AA, Bernstein E, et al.Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells [J]. Genes Dev.2002; 16:948-958.
15. Racz Z, Kaucsar T, Hamar P. The huge world of small RNAs:Regulating networks of microRNAs (review) [J]. Acta Physiol Hung.2011;98:243-251.
16. Hu B, Zhu XL, Fan QX,et al.Experimental study on inhibition of rat ventricular Ikl by RNA interference targeting the KCNJ2 gene [J]. Biosci Trends.2012 Feb;6(1):26-32.
17. Luo HY, Liang HM, Hu XW, et al.Expression of Kir2.1, SCN5a and SCN1b channel genes in mouse cardiomyocytes with various electric properties:patch clamp combined with single cell RT-PCR study [J].Sheng Li Xue Bao.2012 Feb 25;64(1):82-6.
18. Piao L, Li J, McLerie M, et al.Transgenic upregulation of IK1 in the mouse heart is proarrhythmic [J]. Basic Res Cardiol.2007; 102:416-428.
19. Silva J, Rudy Y. Mechanism of pacemaking in I(K1)-downregulated myocytes [J]. Circ Res.2003; 92:261-263.
20. Brendel J, Peukert S. Blockers of the Kv1.5 channel for the treatment of atrial arrhythmias [J]. Curr Med Chem Cardiovasc Hematol Agents 2003; 1:273-287.
21. Kurata Y, Hisatome I, Matsuda H, et al.Dynamical mechanisms of pacemaker generation in IK1-downregulated human ventricular myocytes:insights from bifurcation analyses of a mathematical model [J]. Biophys J 2005;89: 2865-2887.
22. Nakamura TY, Artman M, Rudy B, et al.Inhibition of rat ventricular IK1 with antisense oligonucleotides targeted to Kir2.1 mRNA[J]. Am J Physiol.1998;274: H892-H900.
23. Lopatin AN, Nichols CG. Inward rectifiers in the heart:an update on I(K1) [J]. J Mol Cell Cardiol.2001;33:625-638.
24. Plaster NM, Tawil R, Tristani-Firouzi M,et al.Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome [J]. Cell.2001 May 18;105(4):511-9.
25. Tristani-Firouzi M1, Jensen JL, Donaldson MR,et al.Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome) [J]. Heart Rhythm.2012 Aug;9(8):1310-8.
26. Tristani-Firouzi M, Etheridge SP.Kir 2.1 channelopathies:the Andersen-Tawil syndrome [J]. Pflugers Arch.2010 Jul;460(2):289-94.
27. Chan YC, Siu CW, Lau YM, et al.Synergistic effects of inward rectifier (I) and pacemaker (I) currents on the induction of bioengineered cardiac automaticity[J]. J Cardiovasc Electrophysiol.2009 Sep;20(9):1048-54.
28. Kapoor N, Liang W, Marban E, et al.Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18 [J].Nat Biotechnol. 2013 Jan;31(1):54-62.
29. Wang JC, Liu XY, Lu WL, el al. Pharmacokinetics of intravenously administered stealth liposomal doxombicin modulated with verapamil in rats[J]. Eur J Phann Biopham.2006; 62(1):44-51.
30. DeWitt C, Waksman J. Pharmacology, pathophysiology and management of calcium channel blocker and beta-blocker toxicity[J]. Pharmacology.2004:23(4): 223-38.
31. Huang SK, Nharati S, Graham AR, et al. Chronic incomplete atrioventricular block induced by radiofrequeney catheter ablation [J]. Circulation,1989,80(4): 951-961.
32. Kuck KH, Schluter M, Geiger M, et al. radiofrequeney current catheter ablation of accessory atrioventricular pathways[J]. Lancet,1991,337(8757):1557-1561
33. Kenny JE, Berberian G, Rabkin DG, et al. Ethanol induction of complete heart block in swine[J]. J Surg Res,2006,132(1):142-146.
34. Suto F, Cahili SA, Wilson GJ, et al. A novel rabbit model of variably compensated complete heart bloek[J]. J Appl Physiol,2002,92(3):1199-1204.
35. Lee RJ,Sievers RE,Gallinghouse GJ,et al.Development of a model of complete heart block in rats [J]. J. Appl. Physiol.1998,85:758-763.
36. Miki Yokokawa, Shunsuke Ohnishi,Hatsue Ishibashi-Ueda,et al. Transplantation of Mesenchymal Stem Cells Improves Atrioventricular Conduction in a Rat Model of Complete Atrioventricular Block [J]. Cell Transplantation,2008, Vol. 17,1145-1155.
37. Ishikawa K, Tilemann L, Ladage D, et al.Cardiac gene therapy in large animals: bridge from bench to bedside [J].Gene Ther.2012 Jun;19(6):670-7.
38. Zhang G, Budker V, Wolff JA.High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA [J].Hum Gene Ther. 1999 Jul 1;10(10):1735-7.
39. Stratford-Perricaudet LD, Makeh I, Perricaudet M, et al.Widespread long-term gene transfer to mouse skeletal muscles and heart [J] J Clin Invest.1992 Aug;90(2):626-30.
40. Meluzin J, Vlasin M, Groch L, et al.Intracoronary delivery of bone marrow cells to the acutely infarcted myocardium. Optimization of the delivery technique [J]. Cardiology.2009;112(2):98-106.
41. Cunningham JT, Bruno SB, Higgs KA, et al.Intrapericardial procaine affects volume expansion-induced fos immunoreactivity in unanesthetized rats [J].Exp Neurol.2002 Apr; 174(2):181-92.
42. Magovern CJ, Mack CA, Zhang J, et al.Direct in vivo gene transfer to canine myocardium using a replication-deficient adeno virus vector [J]. Ann Thorac Surg. 1996 Aug;62(2):425-33;discussion 433-4.
43.高文谦,李小鹰。心肌基因转导研究进展[J]。中国老年心脑血管病杂志,2001,3(3)：200-203.
44. Saeed M, Martin A, Jacquier A, et al.Permanent coronary artery occlusion: cardiovascular MR imaging is platform for percutaneous transendocardial delivery and assessment of gene therapy in canine model.Radiology [J].2008 Nov;249(2):560-71.
45. Melnyk P, Zhang L, Shrier A,et al.Differential distribution of Kir2.1 and Kir2.3 subunits in canine atrium and ventricle [J]. Am J Physiol Heart Circ Physiol.2002 Sep; 283(3):H1123-33
46. Dhamoon AS, Pandit SV, Sarmast F,et al. Unique Kir2.x properties determine regional and species differences in the cardiac inward rectifier K+current[J].Circ Res.2004 May 28;94(10):1332-9.
47. Leonoudakis D, Conti LR, Anderson S,et al.Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins [J] J Biol Chem.2004 May 21;279(21):22331-46.
48. Hofherr A, Fakler B, Klocker N.Selective Golgi export of Kir2.1 controls the stoichiometry of functional Kir2.x channel heteromers [J] J Cell Sci.2005 May 1;118(Pt 9):1935-43.
49. Guo D, Ramu Y, Klem AM, et al.Mechanism of rectification in inward-rectifier K+ channels [J] J Gen Physiol.2003 Apr; 121(4):261-75
50. Lu Z.Mechanism of rectification in inward-rectifier K+ channels [J].Annu Rev Physiol.2004;66:103-29.
51. Shin HG, Lu Z.Mechanism of the voltage sensitivity of IRK1 inward-rectifier K+ channel block by the polyamine spermine[J] J Gen Physiol.2005 Apr;125(4):413-26.
52. Sun HY, Xiao GS, Wang Y,et al.Ionic mechanism underlying distinctive excitability in atrium and ventricle of the heart[J].Sheng Li Xue Bao.2014 Feb 25;66(1):85-95.
53. Dhamoon AS, Jalife J.The inward rectifier current (IK1) controls cardiac excitability and is involved in arrhythmogenesis [J].Heart Rhythm.2005 Mar;2(3):316-24.
54. Xia M, Jin Q,et al. A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation.Bendahhou S,Biochem Biophys Res Commun [J].2005 Jul 15;332(4):1012-9.
55. Dobrev D, Wettwer E, Kortner A,et al.Human inward rectifier potassium channels in chronic and postoperative atrial fibrillation[J]. Cardiovasc Res.2002 May;54(2):397-404.
56. Warren M, Guha PK, Berenfeld O,et al.Blockade of the inward rectifying potassium current terminates ventricular fibrillation in the guinea pig heart.J Cardiovasc Electrophysiol [J].2003 Jun; 14(6):621-31.
57. Luo X, Pan Z, Shan H,et al.MicroRNA-26 governs profibrillatory inward-rectifier potassium current changes in atrial fibrillation [J] J Clin Invest.2013 May 1;123(5):1939-51.
58. Girmatsion Z, Biliczki P, Bonauer A,et al.Changes in microRNA-1 expression and IK1 up-regulation in human atrial fibrillation [J]. Heart Rhythm.2009 Dec;6(12):1802-9
59. Liu QH, Li XL, Xu YW,et al.A novel discovery of IK1 channel agonist: zacopride selectively enhances IK1 current and suppresses triggered arrhythmias in the rat [J]. J Cardiovasc Pharmacol.2012 Jan;59(1):37-48.
60. Kokunai Y, Nakata T, Furuta M,et al.A Kir3.4 mutation causes Andersen-Tawil syndrome by an inhibitory effect on Kir2.1 [J]. Neurology.2014 Feb 26.
61. Delannoy E, Sacher F, Maury P,et al.Cardiac characteristics and long-term outcome in Andersen-Tawil syndrome patients related to KCNJ2 mutation [J].Europace.2013 Dec;15(12):1805-11.
62. Marrus SB, Cuculich PS, Wang W, Nerbonne JM.Characterization of a novel, dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome [J]. Channels (Austin).2011 Nov-Dec;5(6):500-9
63. Bendahhou S, Donaldson MR, Plaster NM,et al.Defective potassium channel Kir2.1 trafficking underlies Andersen-Tawil syndrome [J] J Biol Chem.2003 Dec 19;278(51):51779-85.
64. Vaidyanathan R, Vega AL, Song C,et al.The interaction of caveolin 3 protein with the potassium inward rectifier channel Kir2.1:physiology and pathology related to long qt syndrome 9 (LQT9) [J].J Biol Chem.2013 Jun 14;288(24):17472-80.
65. Obeyesekere MN, Klein GJ, Conacher S, Krahn AD.KCNJ2 variant of unknown significance reclassified as long QT syndrome causing ventricular fibrillation [J].Can J Cardiol.2011 Nov-Dec;27(6):870. e11-3.
66. Deo M, Ruan Y, Pandit SV,et al.KCNJ2 mutation in short QT syndrome 3 results in atrial fibrillation and ventricular proarrhythmia [J]. Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):4291-6.
67. Hattori T, Makiyama T, Akao M,et al.A novel gain-of-function KCNJ2 mutation associated with short-QT syndrome impairs inward rectification of Kir2.1 currents [J]. Cardiovasc Res.2012 Mar 15; 93 (4):666-73.
68. El Harchi A, McPate MJ, Zhang Yh,et al.Action potential clamp and chloroquine sensitivity of mutant Kir2.1 channels responsible for variant 3 short QT syndrome [J] J Mol Cell Cardiol.2009 Nov;47(5):743-7.
69. Lange PS, Er F, Gassanov N, Hoppe UC.Andersen mutations of KCNJ2 suppress the native inward rectifier current IK1 in a dominant-negative fashion [J].Cardiovasc Res.2003 Aug 1;59(2):321-7.
70. Priori SG, Pandit SV, Rivolta I,et al.A novel form of short QT syndrome (SQT3) is caused by a mutation in the KCNJ2 gene [J].Circ Res.2005 Apr 15;96(7):800-7.
71. Schulze-Bahr E.Short QT syndrome or Andersen syndrome:Yin and Yang of Kir2.1 channel dysfunction [J].Circ Res.2005 Apr 15;96(7):703-4.
72. Koumi S, Backer CL, Arentzen CE.Characterization of inwardly rectifying K+ channel in human cardiac myocytes. Alterations in channel behavior in myocytes isolated from patients with idiopathic dilated cardiomyopathy [J].Circulation. 1995 Jul15;92(2):164-74.
73. Liu J, Dobrzynski H, Yanni J,et al.Organisation of the mouse sinoatrial node: structure and expression of HCN channels [J]. Cardiovasc Res.2007 Mar 1;73(4):729-38.
74. Karsai A, Muller S, Platz S, et al.Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression [J] Biotechniques.2002 Apr;32(4):790-2,794-6.
75. Dupouey J, Ninove L, Ferrier V,et al.Molecular detection of human rhinoviruses in respiratory samples:a comparison of Taqman probe-, SYBR green I-and BOXTO-based real-time PCR assays [J].Virol J.2014 Feb 18;11 (1):31.
76. Garvey CE, McGowin CL, Foster TP.Development and Evaluation of SYBR Green-I Based Quantitative PCR Assays for Herpes Simplex Virus Type Whole Transcriptome Analysis [J] J Virol Methods.2014 Mar 4.
2. Aiuti A, Roncarolo MG. Ten years of gene therapy for primary immune deficiencies.Hematology Am Soc Hematol Educ Program [J].2009:682-9.
3. Mukherjee S, Thrasher AJ.Gene therapy for PIDs:progress, pitfalls and prospects [J].Gene.2013 Aug 10;525(2):174-81
4. Howe SJ, Mansour MR, Schwarzwaelder K,et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients [J]. J Clin Invest.2008 Sep;118(9):3143-50
5. Weber E, Anderson WF, Kasahara N. Recent advances in retrovirus vector-mediated gene therapy:teaching an old vector new tricks [J]. Curr Opin Mol Ther.2001 Oct;3(5):439-53.
6.杨力明,曲章义,侯咏等。人3型腺病毒六邻体基因表达质粒的构建及表达[J].哈尔滨医科大学学报,2004,38(3)：218-220.
7. Liu L,Pedersen E F. Partial Abstracts of the 3rd Annual Meeting of the China RNA Society & 1st International RNA Workshop in China:SESSION 1: mRNA PROCESSING-Biochemical Identification of Nuclear Proteins p30 Specifically Interacting with Upstream Repressi [J]. Acta Biochimica et Biophysica Sinica,2004,36(2):147.
8. Zhang QY, Jin YZ, Zhang HB, Xie SS.Construction and biological activities of CTLA4Ig adenovirus vectors [J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2003 Jan;19(1):45-8.
9.李方成,李军亮,吴中华等.大鼠GLUT3基因重组腺病毒载体的构建及鉴定[J].中华神经医学杂志,2006,5(11)：1086-1089.
10. Scibetta A G, Copier J, Barrett A, et al. Gene expression changes induced by a recombinant E1/E3 adenovirus type 5 vector in human mammary epithelial cells [J]. Intervirology England,2005,48 (6):350-361.
11. Schowalter D B, Matern D, Vockley J, et al. In vitro correction of medium chain acyl Co A dehydrogenase deficiency with a recombinant adeno viral vector [J] Mol Genet Metab,2005,85(2):88-95.
12.邱峰,陈世知,陈虹.新型腺病毒载体的制备及其特性研究[J]。重庆医科大学学报,2006,31(3)：330-333.
13. Baltimore D, Verma I M, Drost S, et al. Temperature-sensitive DNA polymerase from Rous sarcoma virus mutants [J]. Cancer,1974,34:1-7
14. Esposito D, Craigie R. HIV integrase structure and function [J]. Adv Virus Res, 1999,52:319-333
15. Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector [J]. Science,1996,272: 263-267
16. Mautino MR. Lentiviral vectors for gene therapy of HIV-1 infection [J]. Curr Gene Ther,2002,2(1):23-43
17. Punzon I, Criado L M, Serrano A, et al. Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background [J]. Blood,2004, 103:580-582
18. Jakobsen M, Stenderup K, Rosada C, et al. Amelioration of psoriasis by anti-TNF-alpha RNAi in the xenograft transplantation model [J]. Mol Ther,2009,17: 1743-1753
19.赵世巧。基因治疗的病毒载体研究进展。国外医学临床生物化学与检验学分册2005年10月第26卷第10期。
20. Clare E, Thomas, et al. Progress and problems with the use of viral vectors for gene therapy. Nature review genetics.2003,4:346-358.
21. David Peel. Virus Vectors & Gene Therapy:Problems, Promises & Prospects [J]. MB Ch B Special Study Module Project Report
22. PL Sinn, SL Sauter, PB McCray Jr. Gene Therapy Progress and Prospects: Development of improved lentiviral and retro viral vectors-design, biosafety, and production [J]. Gene Therapy(2005)12,1089-1098.
23. Dann, Christina Tenenhaus. New technology for an old favorite:lentiviral transgenesis and RNAi in rats [J]. Transgenic Research. October 2007,16(5): 571-580.
24. Jakobsson, et al. Efficient transduction of neurons using Ross River glycoprotein pseudotyped lentiviral vectors [J]. Gene Therapy.2006,13(12): 966-973c
25. Logan AC, Lutzko C, Kohn DB. Advances in lentiviral vector design for gene—modification of hematopoietic stem cells [J]. Current Opinion in Biotechnology 2002,13:429-436.
26.周芬,程时远。基因治疗中的非病毒载体[J]。化学通报,2005年,第68卷。
27. Kafri T, Blomer U, Peterson DA, Gage FH, Verma IM. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors [J]. Nat Genet, 1997,17(3):314-317.
28. Kafri T, van Praag H, Gage FH, Verma IM. Lentiviral vectors:regulated gene expression[J]. Moi Ther,2000,1(6):516-521.
29. Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long term expression of the transgene in adult rat brains injected with a lentiviral vector [J]. Proc Natl Acad Sci USA,1996,93(21): 11382-11388.
30. Matrai J, Chuah MK, VandenDriessche T. Recent advances in lentiviral vector development and applications [J]. Mol Ther.2010 Mar;18(3):477-90.
31. Blesch A. Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer [J]. Methods,2004,33(2):164-172.
32. Page K A, Landau N R, Littman D R. Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity [J]. J Virol,1990, 64:5270-5276.
33. Liu B, Paton JF, Kasparov S.Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use in vitro and in vivo.BMC Biotechnol.2008 May 16;8-49.
34. Emery DW.The use of chromatin insulators to improve the expression and safety of integrating gene transfer vectors [J].Hum Gene Ther. 2011 Jun;22(6):761-74.
35. Dropulic B.Lentiviral vectors:their molecular design, safety, and use in laboratory and preclinical research [J].Hum Gene Ther.2011 Jun;22(6):649-57.
36. Delenda C, Gaillard C. Real-time quantitative PGR for the design of lentiviral vector analytical assays [J]. Gene therapy 2005;12 Suppl 1:S36-50.
37. Geraerts M, Willems S, Baekelandt V et al. Comparison of lentiviral vector titration methods [J]. BMC biotechnology 2006; 6:34.
38. Chan C S, Weeber E J, Kurup S, et al Integrin requirement for hippocampal synaptic plasticity and spatial memory [J]. J Neurosci,2003,23(18):7107-7116
1. Marban E, Cho HC. Creation of a biological pacemaker by gene-or cell-based approaches [J]. Med Biol Eng Comput.2007; 45:133-144.
2. Hauser RG, Hayes DL, Kallinen LM, et al.Clinical experience with pacemaker pulse generators and transvenous leads:an 8-year prospective multicenter study [J]. Heart Rhythm 2007;4:154-160.
3. Senaratne J, Irwin ME, Senaratne MP. Pacemaker longevity:are we getting what we are promised [J]? Pacing Clin Electrophysiol 2006; 29:1044-1054.
4. Kay GN, Brinker JA, Kawanishi DT, et al. Risks of spontaneous injury and extraction of an active fixation pacemaker lead:report of the Accufix MulticenterClinical Study and Worldwide Registry [J]. Circulation 1999; 100: 2344-2352.
5. Burney K1, Burchard F, Papouchado M, et al.Cardiac pacing systems and implantable cardiac defibrillators (ICDs):a radiological perspective of equipment, anatomy and complications [J]. Clin Radiol.2004 Aug;59(8):699-708.
6. Friedman RA, Fenrich AL, Kertesz NJ. Congenital complete atrioventricular block [J].Pacing Clin Electrophysiol 2001;24:1681-1688.
7. Greene D, Kang S, Kosenko A, et al.Adrenergic regulation of HCN4 channel requires protein association with β2-adrenergic receptor [J] J Biol Chem.2012 Jul 6;287(28):23690-7.
8. Edelberg JM, Huang DT, Josephson ME, et al.Molecular enhancement of porcine cardiac chronotropy [J]. Heart.2001; 86:559-562.
9. Boink GJ, Duan L, Nearing BD, et al.HCN2/SkMl gene transfer into canine left bundle branch induces stable, autonomically responsive biological pacing at physiological heart rates [J] J Am Coll Cardiol.2013 Mar 19;61(11):1192-201.
10. Plotnikov AN, Sosunov EA, Qu J,et al.Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates [J]. Circulation 2004; 109:506-512.
11. Qu J, Barbuti A, Protas L, et al.HCN2 overexpression in newborn and adult ventricular myocytes:Distinct effects on gating and excitability [J].Circ Res. 2001;89:E8-E14.
12. Miake J, Marban E, Nuss HB. Biological pacemaker created by gene transfer [J]. Nature 2002; 419:132-133.
13. Miake J, Marban E, Nuss HB. Functional role of inward rectifier current in heart probed by Kir2.1 overexpression and dominant-negative suppression [J]. J Clin Invest.2003;111:1529-1536.
14. Paddison PJ, Caudy AA, Bernstein E, et al.Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells [J]. Genes Dev.2002; 16:948-958.
15. Racz Z, Kaucsar T, Hamar P. The huge world of small RNAs:Regulating networks of microRNAs (review) [J]. Acta Physiol Hung.2011;98:243-251.
16. Hu B, Zhu XL, Fan QX,et al.Experimental study on inhibition of rat ventricular Ikl by RNA interference targeting the KCNJ2 gene [J]. Biosci Trends.2012 Feb;6(1):26-32.
17. Luo HY, Liang HM, Hu XW, et al.Expression of Kir2.1, SCN5a and SCN1b channel genes in mouse cardiomyocytes with various electric properties:patch clamp combined with single cell RT-PCR study [J].Sheng Li Xue Bao.2012 Feb 25;64(1):82-6.
18. Piao L, Li J, McLerie M, et al.Transgenic upregulation of IK1 in the mouse heart is proarrhythmic [J]. Basic Res Cardiol.2007; 102:416-428.
19. Silva J, Rudy Y. Mechanism of pacemaking in I(K1)-downregulated myocytes [J]. Circ Res.2003; 92:261-263.
20. Brendel J, Peukert S. Blockers of the Kv1.5 channel for the treatment of atrial arrhythmias [J]. Curr Med Chem Cardiovasc Hematol Agents 2003; 1:273-287.
21. Kurata Y, Hisatome I, Matsuda H, et al.Dynamical mechanisms of pacemaker generation in IK1-downregulated human ventricular myocytes:insights from bifurcation analyses of a mathematical model [J]. Biophys J 2005;89: 2865-2887.
22. Nakamura TY, Artman M, Rudy B, et al.Inhibition of rat ventricular IK1 with antisense oligonucleotides targeted to Kir2.1 mRNA[J]. Am J Physiol.1998;274: H892-H900.
23. Lopatin AN, Nichols CG. Inward rectifiers in the heart:an update on I(K1) [J]. J Mol Cell Cardiol.2001;33:625-638.
24. Plaster NM, Tawil R, Tristani-Firouzi M,et al.Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome [J]. Cell.2001 May 18;105(4):511-9.
25. Tristani-Firouzi M1, Jensen JL, Donaldson MR,et al.Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome) [J]. Heart Rhythm.2012 Aug;9(8):1310-8.
26. Tristani-Firouzi M, Etheridge SP.Kir 2.1 channelopathies:the Andersen-Tawil syndrome [J]. Pflugers Arch.2010 Jul;460(2):289-94.
27. Chan YC, Siu CW, Lau YM, et al.Synergistic effects of inward rectifier (I) and pacemaker (I) currents on the induction of bioengineered cardiac automaticity[J]. J Cardiovasc Electrophysiol.2009 Sep;20(9):1048-54.
28. Kapoor N, Liang W, Marban E, et al.Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18 [J].Nat Biotechnol. 2013 Jan;31(1):54-62.
29. Wang JC, Liu XY, Lu WL, el al. Pharmacokinetics of intravenously administered stealth liposomal doxombicin modulated with verapamil in rats[J]. Eur J Phann Biopham.2006; 62(1):44-51.
30. DeWitt C, Waksman J. Pharmacology, pathophysiology and management of calcium channel blocker and beta-blocker toxicity[J]. Pharmacology.2004:23(4): 223-38.
31. Huang SK, Nharati S, Graham AR, et al. Chronic incomplete atrioventricular block induced by radiofrequeney catheter ablation [J]. Circulation,1989,80(4): 951-961.
32. Kuck KH, Schluter M, Geiger M, et al. radiofrequeney current catheter ablation of accessory atrioventricular pathways[J]. Lancet,1991,337(8757):1557-1561
33. Kenny JE, Berberian G, Rabkin DG, et al. Ethanol induction of complete heart block in swine[J]. J Surg Res,2006,132(1):142-146.
34. Suto F, Cahili SA, Wilson GJ, et al. A novel rabbit model of variably compensated complete heart bloek[J]. J Appl Physiol,2002,92(3):1199-1204.
35. Lee RJ,Sievers RE,Gallinghouse GJ,et al.Development of a model of complete heart block in rats [J]. J. Appl. Physiol.1998,85:758-763.
36. Miki Yokokawa, Shunsuke Ohnishi,Hatsue Ishibashi-Ueda,et al. Transplantation of Mesenchymal Stem Cells Improves Atrioventricular Conduction in a Rat Model of Complete Atrioventricular Block [J]. Cell Transplantation,2008, Vol. 17,1145-1155.
37. Ishikawa K, Tilemann L, Ladage D, et al.Cardiac gene therapy in large animals: bridge from bench to bedside [J].Gene Ther.2012 Jun;19(6):670-7.
38. Zhang G, Budker V, Wolff JA.High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA [J].Hum Gene Ther. 1999 Jul 1;10(10):1735-7.
39. Stratford-Perricaudet LD, Makeh I, Perricaudet M, et al.Widespread long-term gene transfer to mouse skeletal muscles and heart [J] J Clin Invest.1992 Aug;90(2):626-30.
40. Meluzin J, Vlasin M, Groch L, et al.Intracoronary delivery of bone marrow cells to the acutely infarcted myocardium. Optimization of the delivery technique [J]. Cardiology.2009;112(2):98-106.
41. Cunningham JT, Bruno SB, Higgs KA, et al.Intrapericardial procaine affects volume expansion-induced fos immunoreactivity in unanesthetized rats [J].Exp Neurol.2002 Apr; 174(2):181-92.
42. Magovern CJ, Mack CA, Zhang J, et al.Direct in vivo gene transfer to canine myocardium using a replication-deficient adeno virus vector [J]. Ann Thorac Surg. 1996 Aug;62(2):425-33;discussion 433-4.
43.高文谦,李小鹰。心肌基因转导研究进展[J]。中国老年心脑血管病杂志,2001,3(3)：200-203.
44. Saeed M, Martin A, Jacquier A, et al.Permanent coronary artery occlusion: cardiovascular MR imaging is platform for percutaneous transendocardial delivery and assessment of gene therapy in canine model.Radiology [J].2008 Nov;249(2):560-71.
45. Melnyk P, Zhang L, Shrier A,et al.Differential distribution of Kir2.1 and Kir2.3 subunits in canine atrium and ventricle [J]. Am J Physiol Heart Circ Physiol.2002 Sep; 283(3):H1123-33
46. Dhamoon AS, Pandit SV, Sarmast F,et al. Unique Kir2.x properties determine regional and species differences in the cardiac inward rectifier K+current[J].Circ Res.2004 May 28;94(10):1332-9.
47. Leonoudakis D, Conti LR, Anderson S,et al.Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins [J] J Biol Chem.2004 May 21;279(21):22331-46.
48. Hofherr A, Fakler B, Klocker N.Selective Golgi export of Kir2.1 controls the stoichiometry of functional Kir2.x channel heteromers [J] J Cell Sci.2005 May 1;118(Pt 9):1935-43.
49. Guo D, Ramu Y, Klem AM, et al.Mechanism of rectification in inward-rectifier K+ channels [J] J Gen Physiol.2003 Apr; 121(4):261-75
50. Lu Z.Mechanism of rectification in inward-rectifier K+ channels [J].Annu Rev Physiol.2004;66:103-29.
51. Shin HG, Lu Z.Mechanism of the voltage sensitivity of IRK1 inward-rectifier K+ channel block by the polyamine spermine[J] J Gen Physiol.2005 Apr;125(4):413-26.
52. Sun HY, Xiao GS, Wang Y,et al.Ionic mechanism underlying distinctive excitability in atrium and ventricle of the heart[J].Sheng Li Xue Bao.2014 Feb 25;66(1):85-95.
53. Dhamoon AS, Jalife J.The inward rectifier current (IK1) controls cardiac excitability and is involved in arrhythmogenesis [J].Heart Rhythm.2005 Mar;2(3):316-24.
54. Xia M, Jin Q,et al. A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation.Bendahhou S,Biochem Biophys Res Commun [J].2005 Jul 15;332(4):1012-9.
55. Dobrev D, Wettwer E, Kortner A,et al.Human inward rectifier potassium channels in chronic and postoperative atrial fibrillation[J]. Cardiovasc Res.2002 May;54(2):397-404.
56. Warren M, Guha PK, Berenfeld O,et al.Blockade of the inward rectifying potassium current terminates ventricular fibrillation in the guinea pig heart.J Cardiovasc Electrophysiol [J].2003 Jun; 14(6):621-31.
57. Luo X, Pan Z, Shan H,et al.MicroRNA-26 governs profibrillatory inward-rectifier potassium current changes in atrial fibrillation [J] J Clin Invest.2013 May 1;123(5):1939-51.
58. Girmatsion Z, Biliczki P, Bonauer A,et al.Changes in microRNA-1 expression and IK1 up-regulation in human atrial fibrillation [J]. Heart Rhythm.2009 Dec;6(12):1802-9
59. Liu QH, Li XL, Xu YW,et al.A novel discovery of IK1 channel agonist: zacopride selectively enhances IK1 current and suppresses triggered arrhythmias in the rat [J]. J Cardiovasc Pharmacol.2012 Jan;59(1):37-48.
60. Kokunai Y, Nakata T, Furuta M,et al.A Kir3.4 mutation causes Andersen-Tawil syndrome by an inhibitory effect on Kir2.1 [J]. Neurology.2014 Feb 26.
61. Delannoy E, Sacher F, Maury P,et al.Cardiac characteristics and long-term outcome in Andersen-Tawil syndrome patients related to KCNJ2 mutation [J].Europace.2013 Dec;15(12):1805-11.
62. Marrus SB, Cuculich PS, Wang W, Nerbonne JM.Characterization of a novel, dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome [J]. Channels (Austin).2011 Nov-Dec;5(6):500-9
63. Bendahhou S, Donaldson MR, Plaster NM,et al.Defective potassium channel Kir2.1 trafficking underlies Andersen-Tawil syndrome [J] J Biol Chem.2003 Dec 19;278(51):51779-85.
64. Vaidyanathan R, Vega AL, Song C,et al.The interaction of caveolin 3 protein with the potassium inward rectifier channel Kir2.1:physiology and pathology related to long qt syndrome 9 (LQT9) [J].J Biol Chem.2013 Jun 14;288(24):17472-80.
65. Obeyesekere MN, Klein GJ, Conacher S, Krahn AD.KCNJ2 variant of unknown significance reclassified as long QT syndrome causing ventricular fibrillation [J].Can J Cardiol.2011 Nov-Dec;27(6):870. e11-3.
66. Deo M, Ruan Y, Pandit SV,et al.KCNJ2 mutation in short QT syndrome 3 results in atrial fibrillation and ventricular proarrhythmia [J]. Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):4291-6.
67. Hattori T, Makiyama T, Akao M,et al.A novel gain-of-function KCNJ2 mutation associated with short-QT syndrome impairs inward rectification of Kir2.1 currents [J]. Cardiovasc Res.2012 Mar 15; 93 (4):666-73.
68. El Harchi A, McPate MJ, Zhang Yh,et al.Action potential clamp and chloroquine sensitivity of mutant Kir2.1 channels responsible for variant 3 short QT syndrome [J] J Mol Cell Cardiol.2009 Nov;47(5):743-7.
69. Lange PS, Er F, Gassanov N, Hoppe UC.Andersen mutations of KCNJ2 suppress the native inward rectifier current IK1 in a dominant-negative fashion [J].Cardiovasc Res.2003 Aug 1;59(2):321-7.
70. Priori SG, Pandit SV, Rivolta I,et al.A novel form of short QT syndrome (SQT3) is caused by a mutation in the KCNJ2 gene [J].Circ Res.2005 Apr 15;96(7):800-7.
71. Schulze-Bahr E.Short QT syndrome or Andersen syndrome:Yin and Yang of Kir2.1 channel dysfunction [J].Circ Res.2005 Apr 15;96(7):703-4.
72. Koumi S, Backer CL, Arentzen CE.Characterization of inwardly rectifying K+ channel in human cardiac myocytes. Alterations in channel behavior in myocytes isolated from patients with idiopathic dilated cardiomyopathy [J].Circulation. 1995 Jul15;92(2):164-74.
73. Liu J, Dobrzynski H, Yanni J,et al.Organisation of the mouse sinoatrial node: structure and expression of HCN channels [J]. Cardiovasc Res.2007 Mar 1;73(4):729-38.
74. Karsai A, Muller S, Platz S, et al.Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression [J] Biotechniques.2002 Apr;32(4):790-2,794-6.
75. Dupouey J, Ninove L, Ferrier V,et al.Molecular detection of human rhinoviruses in respiratory samples:a comparison of Taqman probe-, SYBR green I-and BOXTO-based real-time PCR assays [J].Virol J.2014 Feb 18;11 (1):31.
76. Garvey CE, McGowin CL, Foster TP.Development and Evaluation of SYBR Green-I Based Quantitative PCR Assays for Herpes Simplex Virus Type Whole Transcriptome Analysis [J] J Virol Methods.2014 Mar 4.