RNA干扰下调骨癌痛大鼠脊髓背角PKCγ表达的镇痛效应研究
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摘要
研究背景
癌性疼痛是晚期癌症患者最常见的症状之一，由于受到生理、情感、认知、行为和社会文化等因素的影响，因而具有很大的易变性和主观性。骨癌痛是一种性质剧烈、烧灼样且伴有严重负性情绪反应的疼痛，常伴有骨折和其他骨骼相关性疾病的发生，并最终导致患者生活质量的严重降低和死亡率的上升。目前对于骨癌痛的治疗已有多种治疗方法，如癌痛的三阶梯治疗方案、放疗、化疗以及手术等，但仍有很大一部分骨癌痛患者其疼痛程度没能得到有效的缓解，同时这些传统的治疗方法往往也伴有大量的副作用，例如阿片类药物容易引起便秘、药物成瘾以及阿片类药物治疗过程中的认知功能障碍等；放疗及化疗过程中在抑制或杀伤癌细胞的同时，对机体内正常细胞也有毒害作用等等，因此极有必要寻求一种基于机制性的、力求能达到高效、作用持久、毒副作用小等优点的新的治疗方案。
脊髓是外周伤害性信息传递及调控的初级中枢之一，尤其是脊髓Ⅱ层，在伤害性信息的传递、整合过程中发挥着重要的作用，观察它们在中枢部位的投射及其所含化学物质的变化，对于阐明伤害性信息的传递和调控机制，具有重要意义。大量的研究结果表明，脊髓背角组织中的蛋白激酶Cγ(protein kinase Cγ，PKCγ)不仅仅参与了脊髓水平伤害性信息的传递与调制，而且促进了脊髓敏化的形成和发展，在慢性疼痛的产生和／或维持中PKCγ可能发挥着重要的作用，因此若能有效降低PKCγ的表达，或许能够获得良好的镇痛效果。
RNA干扰(RNA interference，RNAi)技术是近几年发展起来的转录后基因沉默技术(post-transcriptional gene silencing，PTGS)，它利用小片段双链RNA(double shortRNA，dsRNA)即小干扰RNA(short interfering RNA，siRNA)，特异性降解相应序列的mRNA，从而高效、特异性的沉默相应基因的表达。RNAi作为一种细胞水平的基因敲除工具，业已成为研究内源性基因功能和信号转导途径的有效手段，但如何能持久有效地发挥其降解相应序列mRNA的作用，仍是此技术面临的最大障碍之一。
慢病毒载体是常用的基因转移系统，具有能感染非分裂期细胞和分裂期细胞的特性，一旦病毒与细胞结合，病毒的基因则可整合到细胞的基因组中，成为基因遗传的稳定组成部分，从而可在细胞的分裂过程中传给子代，同时其致病基因已经剔除，重组野生困难，因而用慢病毒载体表达siRNA能安全持久地发挥下调目的基因表达的作用，可用于离体实验和在体实验。
基于以上理论基础，本研究拟通过以下四部分的研究，来评价siRNA干扰下调骨癌痛大鼠脊髓背角组织中PKCγ表达的镇痛效应：1：制作骨癌痛大鼠模型，观察PKCγ在骨癌痛大鼠脊髓背角组织中的表达变化及大鼠疼痛行为学的改变(包括机械缩爪阈值和机械缩爪持续时间)，探讨PKCγ上调值与大鼠疼痛程度改变之间是否具有某种关系，以此初步探讨PKCγ在骨癌痛的产生和／或维持中是否发挥了一定的作用；2：在第一部分研究的基础上，将PKCγ特异性抑制剂GF109203X注入骨癌痛模型大鼠蛛网膜下腔，观察PKCγ功能被抑制后，骨癌痛大鼠疼痛程度是否显著减轻，从而推断PKCγ在骨癌痛发病机制中的作用；3：根据大鼠PKCγ-mRNA序列，设计并构建3个大鼠PKCγ基因的shRNA慢病毒载体，并在体外鉴定其干扰效率，筛选出一个有效的干扰靶点，为下一步试验提供试验基础；4：将有效靶点的慢病毒载体注射到骨癌痛模型大鼠蛛网膜下腔，评价其镇痛效应，为慢性疼痛的转基因治疗和siRNA的在体试验提供理论基础。
研究方法与结果
1．骨癌痛大鼠脊髓背角蛋白激酶Cγ表达的变化及意义
方法：选取成年雌性Wistar大鼠64只，随机分为三组，正常对照组(Na(i|¨)ve组，n=16)：未作任何处理的健康大鼠；假手术组(Sham组，n=16)：大鼠右侧胫骨上段骨髓腔注射热灭活的Walker256细胞10μl；骨癌痛模型组(Cancer-induced bone pain，CIBP组，n=32)：大鼠右侧胫骨上段骨髓腔注入Walker256细胞10μl(4×10~4 cells)制作骨癌痛模型。模型制作前一天及术后21天内每隔3天观察各组大鼠疼痛行为学变化(包括机械缩爪阈值和机械缩爪持续时间)，且各组大鼠分别在术前一天(-1d)及手术后第6、15、21天灌注后取脊髓L4～6节段组织(每个时点随机选择4只)，冰冻切片后作免疫组织化学DAB染色以检测脊髓背角组织中PKCγ的表达；同时对骨癌痛模型组大鼠于术前一天及手术后第6、15、21天，各选4只大鼠取脊髓L4～6节段组织作Western blot分析以检测PKCγ的表达变化，探讨PKCγ上调值与时间相关的大鼠疼痛程度改变之间是否具有某种关系。
结果：术前各组动物基础痛阈值之间无统计学差异(P＞0．05)；CIBP组大鼠术后第6天开始术侧后肢机械缩爪阈值逐渐降低并持续到术后第21天，同期其机械缩爪持续时间明显延长，与Sham组和Na(i|¨)ve组相比，差异有统计学意义(P＜0．05)。免疫组织化学结果显示正常对照组(Na(i|¨)ve组)大鼠L4～6脊髓背角处有少量的PKCγ表达，而骨癌痛模型组(CIBP组)大鼠在术后第6d、15d、21d，其脊髓背角组织中PKCγ表达量明显增多，与假手术组(sham组)相比，差异有统计学意义(P＜0．05)；CIBP组大鼠术后第15天和第21天，其脊髓背角组织中的PKCγ表达量与术后第6天相比亦显著增加，差异有统计学意义(P＜0．05)。Western blot分析结果与免疫组织化学结果基本相一致，即在术后第6d、15d、21d，骨癌痛模型组大鼠脊髓背角组织中PKCγ的表达量与术前相比明显增加，差异有统计学意义(P＜0．05)；相关性分析结果表明脊髓背角组织中PKCγ表达的平均光密度值与骨癌痛大鼠疼痛程度之间具有明显的正相关关系(P＜0．05)。
2．鞘内注射PKCγ抑制剂GF109203X对骨癌痛大鼠痛觉的影响
方法：选取成年雌性Wistar大鼠30只，随机分为五组(每组6只)，对照组(Naive组)：随机选取6只正常大鼠且不做任何手术；骨癌痛模型组(Cancer-induced bone pain，CIBP组)：构建骨癌痛模型后，鞘内不注入任何药物；CIBP+生理盐水组(NormalSaline，NS组)：构建骨癌痛模型后，鞘内注入生理盐水10μL；CIBP+二甲基亚砜组(Dimethylsulfoxide，DMSO组)：构建骨癌痛模型后，鞘内注入DMSO10μL；CIBP+GF109203X(GF109203X组)：构建骨癌痛模型后，鞘内注入PKCγ抑制剂GF109203X10μL。观察各组大鼠注药前及鞘内注药后15、30、45、60、75 min时的疼痛行为学变化(包括机械缩爪阈值和机械缩爪持续时间)；各组大鼠分别在疼痛观察结束后取脊髓L4～6节段组织，采用免疫组织化学方法检测各组大鼠腰段脊髓背角PKCγ的表达变化，探讨PKCγ在骨癌痛发病机制中的作用。
结果：GF109203X组大鼠在鞘内注射GF109203X后其各时点机械缩爪阈值较CIBP组明显升高(P＜0．05)，在注药后60min时其机械缩爪阈值已与正常组相接近，差异无统计学意义(P＞0．05)；GF109203X组大鼠注药后机械缩爪持续时间明显缩短，与CIBP组比较，差异有统计学意义(P＜0．05)。DMSO组大鼠在鞘内注射DMSO后其机械缩爪阈值逐渐升高，同时其机械缩爪持续时间明显缩短，在注药后45、60、75 min时与CIBP组相比，差异有统计学意义(P＜0．05)，但与GF109203X组相比，其升高后的缩爪阈值和缩短的缩爪持续时间仍分别小于GF109203X组相同时点的对应值，且差异有统计学意义(P＜0．05)。免疫组织化学方法检测结果表明：与Naive组相比，其他各组大鼠(CIBP组、NS组、DMSO组和GF109203X组)脊髓背角PKCγ的蛋白表达量均显著增加，差异有统计学意义(P＜0．05)，但DMSO组和GF109203X组大鼠脊髓背角PKCγ的表达量与CIBP组大鼠脊髓背角PKCγ的表达量相比，差异均无统计学意义(P＞0．05)。
3．大鼠PKCγ基因shRNA慢病毒载体的构建及其干扰效率的鉴定
方法：根据PKCγ-mRNA序列，选择3条19nt的靶序列，设计并合成包含正、反义靶序列的互补DNA链，退火后插入到pLVTHM载体的H_1启动子后获得重组质粒(pLV-PKC_1、pLV-PKC_2、pLV-PKC_3)，同时构建一个非特异性对照质粒(pLV-Con)。将所构建的质粒与质粒pMDLg-pRRE、pRsv-REV、pMD2G共转染293T细胞，包装产生慢病毒，再将成功包装的慢病毒分别感染培养的C6细胞，经免疫印迹技术(Western blot)检测PKCγ基因的蛋白表达水平以评价慢病毒载体的抑制效率。
结果：测序证实成功构建了三个大鼠PKCγ基因shRNA慢病毒载体，在分别感染C6细胞后，慢病毒pLV-PKC_2、pLV-PKC_3可明显抑制PKCγ蛋白的表达水平，其中以慢病毒pLV-PKC_2(靶向+1913-+1931位点)的抑制效率最高。
4．鞘内给予PKCγ-shRNA慢病毒载体对骨癌痛大鼠痛觉的影响
方法：选取24只成年雌性Wistar大鼠制作骨癌痛模型，术后第7天经鞘内置管后将模型大鼠随机分为4组：CIBP组(鞘内不注入任何药物)、PBS组(鞘内注入10μl PBS)、pLV-Con组(鞘内注入10μl慢病毒pLV-Con)和pLV-PKC_2组(鞘内注入10μl慢病毒pLV-PKC_2)。于模型制作前，以及模型制作后每隔3天分别测定大鼠机械缩爪阈值和缩爪持续时间；各组大鼠在疼痛观察结束后取脊髓L4～6节段组织，采用免疫组织化学方法、Western blot方法，检测各组大鼠腰段脊髓背角PKCγ的表达变化，探讨慢病毒载体pLV-PKC_2在在体水平能否特异、有效地抑制大鼠PKCγ基因的表达，从而发挥其对骨癌痛大鼠的镇痛效应。
结果：鞘内给药后，PBS组和pLV-Con组大鼠其各个时点的机械缩爪阈值和机械缩爪持续时间与CIBP组同一时点值相比，差异无统计学意义(P＞0．05)；但pLV-PKC_2组大鼠在鞘内注射慢病毒pLV-PKC_2 5天后(即模型制作12天后)，其机械缩爪阈值明显升高、机械缩爪持续时间显著缩短，与CIBP组相比差异有统计学意义(P＜0．05)；免疫组织化学方法和Western blot方法检测结果均显示，经鞘内给药后的PBS组和pLV-Con组大鼠脊髓背角PKCγ蛋白表达量与CIBP组相比，差异无统计学意义(P＞0．05)，但pLV-PKC_2组大鼠脊髓背角PKCγ的蛋白表达量显著降低，与CIBP组相比差异有统计学意义(P＜0．05)。
5．统计学分析
计量资料以均数±标准差((?)±s)表示，采用SPSS 11．0软件进行数据处理，机械缩爪阈值和机械缩爪持续时间采用重复变量数据的方差分析，其余结果比较采用单因素方差分析，以P＜0．05为差异有统计学意义。
研究总结
一、主要研究结果
1．本研究通过疼痛行为学观察和免疫学检测表明，骨癌痛模型大鼠复制成功后，其脊髓背角PKCγ的表达量显著增加，且与疼痛程度的改变之间具有明显的正相关关系。
2．骨癌痛大鼠鞘内注射PKCγ抑制剂GF109203X后，PKCγ的功能被抑制并产生了显著的镇痛效应，说明PKCγ在骨癌痛的产生和／或维持中发挥了重要的作用。
3．根据PKCγ基因mRNA序列，设计并成功构建出3个大鼠PKCγ基因shRNA慢病毒载体，其中慢病毒载体pLV-PKC_2能在蛋白水平上特异、高效地抑制PKCγ基因的表达。
4．骨癌痛大鼠鞘内注射慢病毒pLV-PKC_2后，其脊髓背角PKCγ的表达量显著降低，同时产生了明显的镇痛效应，说明采用转基因镇痛治疗方法能获得确实、有效的镇痛效果。
二、研究结论
在骨癌痛的产生和／或维持中，脊髓背角组织中的PKCγ蛋白参与了疼痛信号的传递；在将成功构建并筛选出的有效慢病毒pLV-PKC_2经鞘内给药后，产生了显著的镇痛效应。
三、创新之处
本研究对骨癌痛的产生机制进行了有益的探索，并针对其产生机制采用了基因镇痛的方法进行治疗：一方面证实了PKCγ在骨癌痛的信号传递过程中起着重要的作用，另一方面利用siRNA技术成功构建了慢病毒载体pLV-PKC_2并在骨癌痛大鼠获得了有效的镇痛作用，说明采用针对骨癌痛发生和／或维持的机制，采用转基因镇痛治疗方法能获得确实、有效的效果，为慢性疼痛的基因治疗和siRNA的在体试验提供了试验基础。
四、展望
1．本研究证实在骨癌痛的产生机制中PKCγ发挥了重要的作用，但在骨癌痛的产生机制中决不是PKCγ单一介导的，还有许多其他信号分子也参与了骨癌痛的产生和／或维持，这需要我们对骨癌痛的产生机制进行进一步深入地研究。
2．RNAi技术是一门新兴的在转录水平上抑制基因表达的技术，已在体内外试验中获得了巨大的成功，但如何将siRNA靶向性导入到目的细胞仍是目前需解决的问题之一。
Pain is one of the common symptoms in terminal cancer patients and the experienceof pain in people with cancer is highly variable and subjective because of severaldimensions,including of physiologic,sensory,affective,cognitive,behavioral,and socio-cultural,etc.Bone cancer pain has been described as a deep,boring sensation that achesand burns and accompanied by episodes of stabbing discomfort.Weight bearing oftenexacerbates this pain.In cancers associated with malignant invasion of the skeleton,metastatic bone destruction leads to increase morbidity and impaired quality of life as aresult of pain,fractures and other skeletal related events.So far,although there are opioid,diphosphonate,radiotherapy,chemotherapy and surgery for relieving cancer pain,it stillreported that a lot of cancer patients have inadequate and undermanaged pain controlbecause of the relative ineffectiveness and the side effects of current treatments.Therefore,it is very necessary for us to explore some novel mechanism-based approaches which havemore effective,long-lasting and less side-effect drugs to relieve the bone cancer pain formost patients. Spinal cord,especially spinal layerⅡ,exerts important functions in the process oftransmitting and integrating nocuity information.It is very important to observe the pulseprojection and the changes of chemical substance in the spinal cord for clarifying themechanism about nocuity information transmission and regulation.Many study resultsmanifestate that PKCγin spinal dorsal horn not only participates nocuity informationtransmission and regulation,but also promotes the formation and development of spinalsensitization.During the development and maintenance of chronic pain,the expression ofPKCγevidently increases and PKCγprobably exerts important functions,so if theexpression quantity of PKCγcan be decreased effectively,perhaps the better analgesiceffect can be obtained.
RNA interference technology (RNAi) is a post-transcriptional gene silencingtechnology (PTGS) which makes use of double short RNA(dsRNA) to specificallydegradate the target mRNA and to highly efficiently silence the corresponding geneexpression.RNAi considered as an effective gene knockout tool for studyingendogenous gene functions and signal pathways has been widespreadly applied toresearch in cell level and animal level,but the biggest obstacle about siRNA applicationis how to carry out the functions of siRNA lastingly and validly.
Lentivirus vectors which are commonly used to transfering gene can infect non-mitotic phase cells and mitotic phase cells.Once lentivirus binds to cells,the gene oflentivirus can integrate to the genome of cells and become a part of stable constituent ofgenetic heredity which can pass down to filial generation during the cell fission process.Because the virulence gene has been rejected and the wild form of lentivirus is verydifficult,the lentivirus vector used to expressing siRNA can safely and lastinglydown-regulate the expression of gene in in-vitro and in-vivo experiments.
Based on above theory,this study was designed four parts to evaluate the analgesiceffect when the expression of PKCγin the spinal dorsal horn was down- regulated bysiRNA interference.
1.After the rat models of cancer-induced bone pain (CIBP) were made,the changes ofPKCγexpression and the changes of nociceptive behaviors was observed,and thenwhether there has some relative relation was explored between the up-regulatedexpressiong of PKCγand the changes of nociceptive behaviors,including mechanicalwithdrawal threshold (MWT) and mechanical withdrawal duration (MWD).
2.In the base of the first study,the CIBP models were intrathecally injected GF109203X(an inhibitor of PKCγ).When the PKCγfunction was inhibited,whether the MWT waslightened and the MWD was significantly prolonged or not were explored.And thenwhether PKCγdefinitly exerts important function in the mechanism of CIBP wasevaluated.
3.After three lentivirus vectors of shRNA based on rat PKCγgene sequence wereconstructed and their interference effects were identified in vitro,a highly effectiveinterference target vector which would provide a basis to next experiment was selected.
4.Based on the third result,the effective target lentivirus vector was injected into ratcavitas subarachnoidealis,the analgesic effect which would provide a theory andexperiment basis to chronic pain gene therapy and siRNA experiment in vivo wasevaluated.
Methods and Results
1:The expression and significance of PKCγin spinal dorsal horn in a ratmodel of cancer bone pain
Methods:Sixty-four female Wistar rats were selected and randomly divided into 3groups:Naive group (not received any surgery,n=16);Sham group (unilateral intra-tibialinjection of 10μl heat-killed cells,n=16) and CIBP group (unilateral intra-tibial injectionwith 10μl Walker 256 carcinoma cells,n=32).The changes of nociceptive behaviors(including MWT and MWD) were recorded on pre-operation and post-operation everyinterval 3 days.After the rats were sacrificed by perfusion with 4% paraform onpost-operation d6~(th),d15~(th) and d21~(st) (4 rats every time point),the lumbar 4-6 spinal cord fordetecting the PKCγexpression changes through immunohistochemistry was removed andsectioned at 30μm on a freezing microtome.Meanwhile the western blot analysis methodwas also used to detect the PKCγexpression of CIBP group on pre-operation andpost-operation(d6~(th)、d15~(th)、d21~(st)).
Results:The basic pain threshold has not any difference among all groups beforeoperation.After operation the MWT of CIBP group gradually decreased and the MWDevidently increased from day 6~(th) to day 21~(st) compared to that of na(?)ve group or sham group(P＜0.05).The immunohistochemistry result displayed that there has a little of PKCγexpression in L4～6 spinal dorsal horn in naive group.The average optical density (AOD) ofPKCγin CIBP group significantly up-regulated compared to sham group on d6~(th)、d15~(th) andd21~(st) (P＜0.05),meanwhile the average optical density(AOD) of PKCγin CIBP group ond15~(th) and d21~(st) was evidently more than that on d6~(th) (P＜0.05).The western blot result wasas same as the immunohistochemistry result.Correlation analysis suggested that there hasobvious positive correlation between the average optical density of PKCγin spinal dorsalhorn and the degree of pain.
2:Analgesic effect of intrathecal injection of PKCγinhibitor GF109203Xin a rat model of bone cancer pain
Methods:Thirty female Wistar rats were selected and randomly divided into 5 groupsas follows (n=6 each):Naive group (normal rats and not received any intervention);CIBPgroup (only unilateral intra-tibial injection with 10μl Walker 256 carcinoma cells);CIBP+Normal Saline (NS group,intrathecal injection of normal saline10μL to the CIBP rats);CIBP + DMSO group (DMSO group,intrathecal injection of DMSO 10μL to the CIBP rats);CIBP +GF109203X (GF109203X group,intrathecal injection of GF109203X 10μL to theCIBP rats).Changes of nociceptive behaviors were assessed by MWT and MWD beforeinjection of drugs as well as 15、30、45、60、75min after injection.After nociceptivebehavior assessments were finished the rats were sacrificed by perfusion with 4% paraform,the lumbar 4-6 spinal cord was removed and sectioned on a freezing microtome,and thenthe expression of PKCγwas detected through immunohistochemistry.
Results:After intrathecal administration of GF109203X,the MWT of GF109203Xgroup was significantly higher than that of CIBP group (P＜0.05),and at 60 min afteradministration the MWT of GF109203X group was closed to the MWT of na(?)ve group(P＞0.05),meanwhile The MWD of GF109203X group evidently shorter than that ofCIBP group (P＜0.05).The MWT of DMSO group significantly increased and the MWDevidently decreased at 45、60、75min after administration compared to that of CIBP group(P＜0.05),but there had still significant differences in MWT and MWD between DMSOgroup and GF109203X group (P＜0.05).The expression levels of PKCγfailed to displaysignificant changes in spinal dorsal horn in the respective intrathecal injection group,including NS group、DMSO group and GF109203X group compared to CIBP group byimmunohistochemistry (P＞0.05).
3:Construction of shRNA lentivirus vector targeting rat PKCγgene andidentification of its interference efficiency
Methods:Three target sequences (19nt) were selected according to the rat PKCγ-mRNA sequence,whereafter the sense and antisense oligonucleotides were designed andsynthesized.After annealing,these double strand DNAs were cloned to the pLVTHMvector which contains H1 promoter and green fluorescent protein (GFP),meanwhile ageneral sequence was used as a negative control.All lentivirus particles produced by 293Tcell transfection with the vector plasmid、pMDLg-pRRE、pRsv-REV and pMD2G wereinfected the C6 cell and the Western blot was used to identify the inhibition efficiency ofthe constructed RNAi lentivirus vectors.
Results:DNA sequencing demonstrated that the shRNA lentivirus vectors wereconstructed successfully.After infected C6 cells,the lentivirus of pLV-PKC_2、pLV-PKC_3could evidently decrease the expression of PKCγ,especially the inhibition efficiency ofpLV-PKC_2 (target site:+1913-+1931)) was the highest.
4:Effect of analgesia by intrathecal injection ofPKCγ-shRNA lentivirus ina rat model of bone cancer pain
Methods:Twenty-four female Wistar rats which had been made CIBP model wererandomly divided into 4 groups as follows (n=6 each):CIBP group (not injection anydrugs);PBS group (intrathecal injection of PBS 10μL);pLV-Con group (intrathecalinjection of lentivirus pLV-Con 10μL);pLV-PKC_2 group (intrathecal injection of lentiviruspLV-PKC_2 10μL).Changes of nociceptive behaviors including MWT and MWD wereassessed on pre-injection and post-injection every interval 3 days.After pain assessmentswere finished,the rats were sacrificed.The lumbar 4-6 spinal cord was removed anddetected the expression of PKCγthrough immunohistochemistry and Western blot.
Results:After intrathecal administration,the MWT and MWD of PBS group andpLV-Con group had no any difference compared to CIBP group,but the MWT ofpLV-PKC_2 group significantly increased and the MWD evidently prolonged compared toCIBP group after 5 days in intrathecal injection of lentivirns pLV-PKC_2 (I.e.12~(nd) day aftermodels were made).The expression levels of PKCγfailed to display evident changes inspinal dorsal horn in PBS group and pLV-Con group compared to CIBP group byImmunohisto-chemistry and Western blot,however the PKCγexpression levels inpLV-PKC_2 group significantly reduced compared to CIBP group (P＜0.05).
5:Statistical analysis
All of the analyses were performed by SPSS 11.0 software package.Measurementdata are presented as mean + standard deviation (SD).Group comparisons were made usinga One-way repeated measures ANOVA (behavioral testing data) and the other results usingANOVA.A value of P＜0.05 was considered statistically significant.
Major Results:
1.Through observing the pain behavioral changes of CIBP rats and the immunochemistrydetection,there has obvious positive correlation between the degree of pain and theexpression of PKCγin spinal dorsal horn.
2.When the PKCγinhibitor GF109203X was intrathecally injected to CIBP rats,it couldbring about significant analgesic effect,which suggested that could exert importantfunction in the development/maintenance of bone cancer pain.
3.The shRNA lentivirus vectors based on PKCγgene sequence were constructedsuccessfully and lentivirus pLV-PKC_2 could specifically,highly efficiently inhibitPKCγgene expression.
4.When lentivirus pLV-PKC_2 was intrathecally injected to CIBP rats,it could generateevident analgesic effect and the expression of PKCγwas significantly down-regulated,which suggested that gene analgesic method mediated by siRNA could obtain definite,effective analgesic effect.
Major Conclusion:
In the development and maintenance of bone cancer pain,PKCγas an importantsignal molecular participates the transmission of nociceptive signal.After the lentiviruspLV-PKC_2 was intrathecally injected,it could bring about significant analgesic effect.
Innovation:
This study is a novel gene therapy based on the mechanism of CIBP.One side,itidentified signal molecular PKCγexerted important functions in the signal transmission ofCIBP;on the other hand,the lentivirus pLV-PKC_2 based on siRNA technology could produce significant analgesic effect,which suggested that gene analgesic therapy methodcould obtain definite,effective analgesic effect,and it could provide theory basis andexperiment basis to chronic pain therapy and the in vivo application of siRNA.
癌性疼痛是晚期癌症患者最常见的症状之一，由于受到生理、情感、认知、行为和社会文化等因素的影响，因而具有很大的易变性和主观性。骨癌痛是一种性质剧烈、烧灼样且伴有严重负性情绪反应的疼痛，常伴有骨折和其他骨骼相关性疾病的发生，并最终导致患者生活质量的严重降低和死亡率的上升。目前对于骨癌痛的治疗已有多种治疗方法，如癌痛的三阶梯治疗方案、放疗、化疗以及手术等，但仍有很大一部分骨癌痛患者其疼痛程度没能得到有效的缓解，同时这些传统的治疗方法往往也伴有大量的副作用，例如阿片类药物容易引起便秘、药物成瘾以及阿片类药物治疗过程中的认知功能障碍等；放疗及化疗过程中在抑制或杀伤癌细胞的同时，对机体内正常细胞也有毒害作用等等，因此极有必要寻求一种基于机制性的、力求能达到高效、作用持久、毒副作用小等优点的新的治疗方案。
脊髓是外周伤害性信息传递及调控的初级中枢之一，尤其是脊髓Ⅱ层，在伤害性信息的传递、整合过程中发挥着重要的作用，观察它们在中枢部位的投射及其所含化学物质的变化，对于阐明伤害性信息的传递和调控机制，具有重要意义。大量的研究结果表明，脊髓背角组织中的蛋白激酶Cγ(protein kinase Cγ，PKCγ)不仅仅参与了脊髓水平伤害性信息的传递与调制，而且促进了脊髓敏化的形成和发展，在慢性疼痛的产生和／或维持中PKCγ可能发挥着重要的作用，因此若能有效降低PKCγ的表达，或许能够获得良好的镇痛效果。
RNA干扰(RNA interference，RNAi)技术是近几年发展起来的转录后基因沉默技术(post-transcriptional gene silencing，PTGS)，它利用小片段双链RNA(double shortRNA，dsRNA)即小干扰RNA(short interfering RNA，siRNA)，特异性降解相应序列的mRNA，从而高效、特异性的沉默相应基因的表达。RNAi作为一种细胞水平的基因敲除工具，业已成为研究内源性基因功能和信号转导途径的有效手段，但如何能持久有效地发挥其降解相应序列mRNA的作用，仍是此技术面临的最大障碍之一。
慢病毒载体是常用的基因转移系统，具有能感染非分裂期细胞和分裂期细胞的特性，一旦病毒与细胞结合，病毒的基因则可整合到细胞的基因组中，成为基因遗传的稳定组成部分，从而可在细胞的分裂过程中传给子代，同时其致病基因已经剔除，重组野生困难，因而用慢病毒载体表达siRNA能安全持久地发挥下调目的基因表达的作用，可用于离体实验和在体实验。
基于以上理论基础，本研究拟通过以下四部分的研究，来评价siRNA干扰下调骨癌痛大鼠脊髓背角组织中PKCγ表达的镇痛效应：1：制作骨癌痛大鼠模型，观察PKCγ在骨癌痛大鼠脊髓背角组织中的表达变化及大鼠疼痛行为学的改变(包括机械缩爪阈值和机械缩爪持续时间)，探讨PKCγ上调值与大鼠疼痛程度改变之间是否具有某种关系，以此初步探讨PKCγ在骨癌痛的产生和／或维持中是否发挥了一定的作用；2：在第一部分研究的基础上，将PKCγ特异性抑制剂GF109203X注入骨癌痛模型大鼠蛛网膜下腔，观察PKCγ功能被抑制后，骨癌痛大鼠疼痛程度是否显著减轻，从而推断PKCγ在骨癌痛发病机制中的作用；3：根据大鼠PKCγ-mRNA序列，设计并构建3个大鼠PKCγ基因的shRNA慢病毒载体，并在体外鉴定其干扰效率，筛选出一个有效的干扰靶点，为下一步试验提供试验基础；4：将有效靶点的慢病毒载体注射到骨癌痛模型大鼠蛛网膜下腔，评价其镇痛效应，为慢性疼痛的转基因治疗和siRNA的在体试验提供理论基础。
研究方法与结果
1．骨癌痛大鼠脊髓背角蛋白激酶Cγ表达的变化及意义
方法：选取成年雌性Wistar大鼠64只，随机分为三组，正常对照组(Na(i|¨)ve组，n=16)：未作任何处理的健康大鼠；假手术组(Sham组，n=16)：大鼠右侧胫骨上段骨髓腔注射热灭活的Walker256细胞10μl；骨癌痛模型组(Cancer-induced bone pain，CIBP组，n=32)：大鼠右侧胫骨上段骨髓腔注入Walker256细胞10μl(4×10~4 cells)制作骨癌痛模型。模型制作前一天及术后21天内每隔3天观察各组大鼠疼痛行为学变化(包括机械缩爪阈值和机械缩爪持续时间)，且各组大鼠分别在术前一天(-1d)及手术后第6、15、21天灌注后取脊髓L4～6节段组织(每个时点随机选择4只)，冰冻切片后作免疫组织化学DAB染色以检测脊髓背角组织中PKCγ的表达；同时对骨癌痛模型组大鼠于术前一天及手术后第6、15、21天，各选4只大鼠取脊髓L4～6节段组织作Western blot分析以检测PKCγ的表达变化，探讨PKCγ上调值与时间相关的大鼠疼痛程度改变之间是否具有某种关系。
结果：术前各组动物基础痛阈值之间无统计学差异(P＞0．05)；CIBP组大鼠术后第6天开始术侧后肢机械缩爪阈值逐渐降低并持续到术后第21天，同期其机械缩爪持续时间明显延长，与Sham组和Na(i|¨)ve组相比，差异有统计学意义(P＜0．05)。免疫组织化学结果显示正常对照组(Na(i|¨)ve组)大鼠L4～6脊髓背角处有少量的PKCγ表达，而骨癌痛模型组(CIBP组)大鼠在术后第6d、15d、21d，其脊髓背角组织中PKCγ表达量明显增多，与假手术组(sham组)相比，差异有统计学意义(P＜0．05)；CIBP组大鼠术后第15天和第21天，其脊髓背角组织中的PKCγ表达量与术后第6天相比亦显著增加，差异有统计学意义(P＜0．05)。Western blot分析结果与免疫组织化学结果基本相一致，即在术后第6d、15d、21d，骨癌痛模型组大鼠脊髓背角组织中PKCγ的表达量与术前相比明显增加，差异有统计学意义(P＜0．05)；相关性分析结果表明脊髓背角组织中PKCγ表达的平均光密度值与骨癌痛大鼠疼痛程度之间具有明显的正相关关系(P＜0．05)。
2．鞘内注射PKCγ抑制剂GF109203X对骨癌痛大鼠痛觉的影响
方法：选取成年雌性Wistar大鼠30只，随机分为五组(每组6只)，对照组(Naive组)：随机选取6只正常大鼠且不做任何手术；骨癌痛模型组(Cancer-induced bone pain，CIBP组)：构建骨癌痛模型后，鞘内不注入任何药物；CIBP+生理盐水组(NormalSaline，NS组)：构建骨癌痛模型后，鞘内注入生理盐水10μL；CIBP+二甲基亚砜组(Dimethylsulfoxide，DMSO组)：构建骨癌痛模型后，鞘内注入DMSO10μL；CIBP+GF109203X(GF109203X组)：构建骨癌痛模型后，鞘内注入PKCγ抑制剂GF109203X10μL。观察各组大鼠注药前及鞘内注药后15、30、45、60、75 min时的疼痛行为学变化(包括机械缩爪阈值和机械缩爪持续时间)；各组大鼠分别在疼痛观察结束后取脊髓L4～6节段组织，采用免疫组织化学方法检测各组大鼠腰段脊髓背角PKCγ的表达变化，探讨PKCγ在骨癌痛发病机制中的作用。
结果：GF109203X组大鼠在鞘内注射GF109203X后其各时点机械缩爪阈值较CIBP组明显升高(P＜0．05)，在注药后60min时其机械缩爪阈值已与正常组相接近，差异无统计学意义(P＞0．05)；GF109203X组大鼠注药后机械缩爪持续时间明显缩短，与CIBP组比较，差异有统计学意义(P＜0．05)。DMSO组大鼠在鞘内注射DMSO后其机械缩爪阈值逐渐升高，同时其机械缩爪持续时间明显缩短，在注药后45、60、75 min时与CIBP组相比，差异有统计学意义(P＜0．05)，但与GF109203X组相比，其升高后的缩爪阈值和缩短的缩爪持续时间仍分别小于GF109203X组相同时点的对应值，且差异有统计学意义(P＜0．05)。免疫组织化学方法检测结果表明：与Naive组相比，其他各组大鼠(CIBP组、NS组、DMSO组和GF109203X组)脊髓背角PKCγ的蛋白表达量均显著增加，差异有统计学意义(P＜0．05)，但DMSO组和GF109203X组大鼠脊髓背角PKCγ的表达量与CIBP组大鼠脊髓背角PKCγ的表达量相比，差异均无统计学意义(P＞0．05)。
3．大鼠PKCγ基因shRNA慢病毒载体的构建及其干扰效率的鉴定
方法：根据PKCγ-mRNA序列，选择3条19nt的靶序列，设计并合成包含正、反义靶序列的互补DNA链，退火后插入到pLVTHM载体的H_1启动子后获得重组质粒(pLV-PKC_1、pLV-PKC_2、pLV-PKC_3)，同时构建一个非特异性对照质粒(pLV-Con)。将所构建的质粒与质粒pMDLg-pRRE、pRsv-REV、pMD2G共转染293T细胞，包装产生慢病毒，再将成功包装的慢病毒分别感染培养的C6细胞，经免疫印迹技术(Western blot)检测PKCγ基因的蛋白表达水平以评价慢病毒载体的抑制效率。
结果：测序证实成功构建了三个大鼠PKCγ基因shRNA慢病毒载体，在分别感染C6细胞后，慢病毒pLV-PKC_2、pLV-PKC_3可明显抑制PKCγ蛋白的表达水平，其中以慢病毒pLV-PKC_2(靶向+1913-+1931位点)的抑制效率最高。
4．鞘内给予PKCγ-shRNA慢病毒载体对骨癌痛大鼠痛觉的影响
方法：选取24只成年雌性Wistar大鼠制作骨癌痛模型，术后第7天经鞘内置管后将模型大鼠随机分为4组：CIBP组(鞘内不注入任何药物)、PBS组(鞘内注入10μl PBS)、pLV-Con组(鞘内注入10μl慢病毒pLV-Con)和pLV-PKC_2组(鞘内注入10μl慢病毒pLV-PKC_2)。于模型制作前，以及模型制作后每隔3天分别测定大鼠机械缩爪阈值和缩爪持续时间；各组大鼠在疼痛观察结束后取脊髓L4～6节段组织，采用免疫组织化学方法、Western blot方法，检测各组大鼠腰段脊髓背角PKCγ的表达变化，探讨慢病毒载体pLV-PKC_2在在体水平能否特异、有效地抑制大鼠PKCγ基因的表达，从而发挥其对骨癌痛大鼠的镇痛效应。
结果：鞘内给药后，PBS组和pLV-Con组大鼠其各个时点的机械缩爪阈值和机械缩爪持续时间与CIBP组同一时点值相比，差异无统计学意义(P＞0．05)；但pLV-PKC_2组大鼠在鞘内注射慢病毒pLV-PKC_2 5天后(即模型制作12天后)，其机械缩爪阈值明显升高、机械缩爪持续时间显著缩短，与CIBP组相比差异有统计学意义(P＜0．05)；免疫组织化学方法和Western blot方法检测结果均显示，经鞘内给药后的PBS组和pLV-Con组大鼠脊髓背角PKCγ蛋白表达量与CIBP组相比，差异无统计学意义(P＞0．05)，但pLV-PKC_2组大鼠脊髓背角PKCγ的蛋白表达量显著降低，与CIBP组相比差异有统计学意义(P＜0．05)。
5．统计学分析
计量资料以均数±标准差((?)±s)表示，采用SPSS 11．0软件进行数据处理，机械缩爪阈值和机械缩爪持续时间采用重复变量数据的方差分析，其余结果比较采用单因素方差分析，以P＜0．05为差异有统计学意义。
研究总结
一、主要研究结果
1．本研究通过疼痛行为学观察和免疫学检测表明，骨癌痛模型大鼠复制成功后，其脊髓背角PKCγ的表达量显著增加，且与疼痛程度的改变之间具有明显的正相关关系。
2．骨癌痛大鼠鞘内注射PKCγ抑制剂GF109203X后，PKCγ的功能被抑制并产生了显著的镇痛效应，说明PKCγ在骨癌痛的产生和／或维持中发挥了重要的作用。
3．根据PKCγ基因mRNA序列，设计并成功构建出3个大鼠PKCγ基因shRNA慢病毒载体，其中慢病毒载体pLV-PKC_2能在蛋白水平上特异、高效地抑制PKCγ基因的表达。
4．骨癌痛大鼠鞘内注射慢病毒pLV-PKC_2后，其脊髓背角PKCγ的表达量显著降低，同时产生了明显的镇痛效应，说明采用转基因镇痛治疗方法能获得确实、有效的镇痛效果。
二、研究结论
在骨癌痛的产生和／或维持中，脊髓背角组织中的PKCγ蛋白参与了疼痛信号的传递；在将成功构建并筛选出的有效慢病毒pLV-PKC_2经鞘内给药后，产生了显著的镇痛效应。
三、创新之处
本研究对骨癌痛的产生机制进行了有益的探索，并针对其产生机制采用了基因镇痛的方法进行治疗：一方面证实了PKCγ在骨癌痛的信号传递过程中起着重要的作用，另一方面利用siRNA技术成功构建了慢病毒载体pLV-PKC_2并在骨癌痛大鼠获得了有效的镇痛作用，说明采用针对骨癌痛发生和／或维持的机制，采用转基因镇痛治疗方法能获得确实、有效的效果，为慢性疼痛的基因治疗和siRNA的在体试验提供了试验基础。
四、展望
1．本研究证实在骨癌痛的产生机制中PKCγ发挥了重要的作用，但在骨癌痛的产生机制中决不是PKCγ单一介导的，还有许多其他信号分子也参与了骨癌痛的产生和／或维持，这需要我们对骨癌痛的产生机制进行进一步深入地研究。
2．RNAi技术是一门新兴的在转录水平上抑制基因表达的技术，已在体内外试验中获得了巨大的成功，但如何将siRNA靶向性导入到目的细胞仍是目前需解决的问题之一。
Pain is one of the common symptoms in terminal cancer patients and the experienceof pain in people with cancer is highly variable and subjective because of severaldimensions,including of physiologic,sensory,affective,cognitive,behavioral,and socio-cultural,etc.Bone cancer pain has been described as a deep,boring sensation that achesand burns and accompanied by episodes of stabbing discomfort.Weight bearing oftenexacerbates this pain.In cancers associated with malignant invasion of the skeleton,metastatic bone destruction leads to increase morbidity and impaired quality of life as aresult of pain,fractures and other skeletal related events.So far,although there are opioid,diphosphonate,radiotherapy,chemotherapy and surgery for relieving cancer pain,it stillreported that a lot of cancer patients have inadequate and undermanaged pain controlbecause of the relative ineffectiveness and the side effects of current treatments.Therefore,it is very necessary for us to explore some novel mechanism-based approaches which havemore effective,long-lasting and less side-effect drugs to relieve the bone cancer pain formost patients. Spinal cord,especially spinal layerⅡ,exerts important functions in the process oftransmitting and integrating nocuity information.It is very important to observe the pulseprojection and the changes of chemical substance in the spinal cord for clarifying themechanism about nocuity information transmission and regulation.Many study resultsmanifestate that PKCγin spinal dorsal horn not only participates nocuity informationtransmission and regulation,but also promotes the formation and development of spinalsensitization.During the development and maintenance of chronic pain,the expression ofPKCγevidently increases and PKCγprobably exerts important functions,so if theexpression quantity of PKCγcan be decreased effectively,perhaps the better analgesiceffect can be obtained.
RNA interference technology (RNAi) is a post-transcriptional gene silencingtechnology (PTGS) which makes use of double short RNA(dsRNA) to specificallydegradate the target mRNA and to highly efficiently silence the corresponding geneexpression.RNAi considered as an effective gene knockout tool for studyingendogenous gene functions and signal pathways has been widespreadly applied toresearch in cell level and animal level,but the biggest obstacle about siRNA applicationis how to carry out the functions of siRNA lastingly and validly.
Lentivirus vectors which are commonly used to transfering gene can infect non-mitotic phase cells and mitotic phase cells.Once lentivirus binds to cells,the gene oflentivirus can integrate to the genome of cells and become a part of stable constituent ofgenetic heredity which can pass down to filial generation during the cell fission process.Because the virulence gene has been rejected and the wild form of lentivirus is verydifficult,the lentivirus vector used to expressing siRNA can safely and lastinglydown-regulate the expression of gene in in-vitro and in-vivo experiments.
Based on above theory,this study was designed four parts to evaluate the analgesiceffect when the expression of PKCγin the spinal dorsal horn was down- regulated bysiRNA interference.
1.After the rat models of cancer-induced bone pain (CIBP) were made,the changes ofPKCγexpression and the changes of nociceptive behaviors was observed,and thenwhether there has some relative relation was explored between the up-regulatedexpressiong of PKCγand the changes of nociceptive behaviors,including mechanicalwithdrawal threshold (MWT) and mechanical withdrawal duration (MWD).
2.In the base of the first study,the CIBP models were intrathecally injected GF109203X(an inhibitor of PKCγ).When the PKCγfunction was inhibited,whether the MWT waslightened and the MWD was significantly prolonged or not were explored.And thenwhether PKCγdefinitly exerts important function in the mechanism of CIBP wasevaluated.
3.After three lentivirus vectors of shRNA based on rat PKCγgene sequence wereconstructed and their interference effects were identified in vitro,a highly effectiveinterference target vector which would provide a basis to next experiment was selected.
4.Based on the third result,the effective target lentivirus vector was injected into ratcavitas subarachnoidealis,the analgesic effect which would provide a theory andexperiment basis to chronic pain gene therapy and siRNA experiment in vivo wasevaluated.
Methods and Results
1:The expression and significance of PKCγin spinal dorsal horn in a ratmodel of cancer bone pain
Methods:Sixty-four female Wistar rats were selected and randomly divided into 3groups:Naive group (not received any surgery,n=16);Sham group (unilateral intra-tibialinjection of 10μl heat-killed cells,n=16) and CIBP group (unilateral intra-tibial injectionwith 10μl Walker 256 carcinoma cells,n=32).The changes of nociceptive behaviors(including MWT and MWD) were recorded on pre-operation and post-operation everyinterval 3 days.After the rats were sacrificed by perfusion with 4% paraform onpost-operation d6~(th),d15~(th) and d21~(st) (4 rats every time point),the lumbar 4-6 spinal cord fordetecting the PKCγexpression changes through immunohistochemistry was removed andsectioned at 30μm on a freezing microtome.Meanwhile the western blot analysis methodwas also used to detect the PKCγexpression of CIBP group on pre-operation andpost-operation(d6~(th)、d15~(th)、d21~(st)).
Results:The basic pain threshold has not any difference among all groups beforeoperation.After operation the MWT of CIBP group gradually decreased and the MWDevidently increased from day 6~(th) to day 21~(st) compared to that of na(?)ve group or sham group(P＜0.05).The immunohistochemistry result displayed that there has a little of PKCγexpression in L4～6 spinal dorsal horn in naive group.The average optical density (AOD) ofPKCγin CIBP group significantly up-regulated compared to sham group on d6~(th)、d15~(th) andd21~(st) (P＜0.05),meanwhile the average optical density(AOD) of PKCγin CIBP group ond15~(th) and d21~(st) was evidently more than that on d6~(th) (P＜0.05).The western blot result wasas same as the immunohistochemistry result.Correlation analysis suggested that there hasobvious positive correlation between the average optical density of PKCγin spinal dorsalhorn and the degree of pain.
2:Analgesic effect of intrathecal injection of PKCγinhibitor GF109203Xin a rat model of bone cancer pain
Methods:Thirty female Wistar rats were selected and randomly divided into 5 groupsas follows (n=6 each):Naive group (normal rats and not received any intervention);CIBPgroup (only unilateral intra-tibial injection with 10μl Walker 256 carcinoma cells);CIBP+Normal Saline (NS group,intrathecal injection of normal saline10μL to the CIBP rats);CIBP + DMSO group (DMSO group,intrathecal injection of DMSO 10μL to the CIBP rats);CIBP +GF109203X (GF109203X group,intrathecal injection of GF109203X 10μL to theCIBP rats).Changes of nociceptive behaviors were assessed by MWT and MWD beforeinjection of drugs as well as 15、30、45、60、75min after injection.After nociceptivebehavior assessments were finished the rats were sacrificed by perfusion with 4% paraform,the lumbar 4-6 spinal cord was removed and sectioned on a freezing microtome,and thenthe expression of PKCγwas detected through immunohistochemistry.
Results:After intrathecal administration of GF109203X,the MWT of GF109203Xgroup was significantly higher than that of CIBP group (P＜0.05),and at 60 min afteradministration the MWT of GF109203X group was closed to the MWT of na(?)ve group(P＞0.05),meanwhile The MWD of GF109203X group evidently shorter than that ofCIBP group (P＜0.05).The MWT of DMSO group significantly increased and the MWDevidently decreased at 45、60、75min after administration compared to that of CIBP group(P＜0.05),but there had still significant differences in MWT and MWD between DMSOgroup and GF109203X group (P＜0.05).The expression levels of PKCγfailed to displaysignificant changes in spinal dorsal horn in the respective intrathecal injection group,including NS group、DMSO group and GF109203X group compared to CIBP group byimmunohistochemistry (P＞0.05).
3:Construction of shRNA lentivirus vector targeting rat PKCγgene andidentification of its interference efficiency
Methods:Three target sequences (19nt) were selected according to the rat PKCγ-mRNA sequence,whereafter the sense and antisense oligonucleotides were designed andsynthesized.After annealing,these double strand DNAs were cloned to the pLVTHMvector which contains H1 promoter and green fluorescent protein (GFP),meanwhile ageneral sequence was used as a negative control.All lentivirus particles produced by 293Tcell transfection with the vector plasmid、pMDLg-pRRE、pRsv-REV and pMD2G wereinfected the C6 cell and the Western blot was used to identify the inhibition efficiency ofthe constructed RNAi lentivirus vectors.
Results:DNA sequencing demonstrated that the shRNA lentivirus vectors wereconstructed successfully.After infected C6 cells,the lentivirus of pLV-PKC_2、pLV-PKC_3could evidently decrease the expression of PKCγ,especially the inhibition efficiency ofpLV-PKC_2 (target site:+1913-+1931)) was the highest.
4:Effect of analgesia by intrathecal injection ofPKCγ-shRNA lentivirus ina rat model of bone cancer pain
Methods:Twenty-four female Wistar rats which had been made CIBP model wererandomly divided into 4 groups as follows (n=6 each):CIBP group (not injection anydrugs);PBS group (intrathecal injection of PBS 10μL);pLV-Con group (intrathecalinjection of lentivirus pLV-Con 10μL);pLV-PKC_2 group (intrathecal injection of lentiviruspLV-PKC_2 10μL).Changes of nociceptive behaviors including MWT and MWD wereassessed on pre-injection and post-injection every interval 3 days.After pain assessmentswere finished,the rats were sacrificed.The lumbar 4-6 spinal cord was removed anddetected the expression of PKCγthrough immunohistochemistry and Western blot.
Results:After intrathecal administration,the MWT and MWD of PBS group andpLV-Con group had no any difference compared to CIBP group,but the MWT ofpLV-PKC_2 group significantly increased and the MWD evidently prolonged compared toCIBP group after 5 days in intrathecal injection of lentivirns pLV-PKC_2 (I.e.12~(nd) day aftermodels were made).The expression levels of PKCγfailed to display evident changes inspinal dorsal horn in PBS group and pLV-Con group compared to CIBP group byImmunohisto-chemistry and Western blot,however the PKCγexpression levels inpLV-PKC_2 group significantly reduced compared to CIBP group (P＜0.05).
5:Statistical analysis
All of the analyses were performed by SPSS 11.0 software package.Measurementdata are presented as mean + standard deviation (SD).Group comparisons were made usinga One-way repeated measures ANOVA (behavioral testing data) and the other results usingANOVA.A value of P＜0.05 was considered statistically significant.
Major Results:
1.Through observing the pain behavioral changes of CIBP rats and the immunochemistrydetection,there has obvious positive correlation between the degree of pain and theexpression of PKCγin spinal dorsal horn.
2.When the PKCγinhibitor GF109203X was intrathecally injected to CIBP rats,it couldbring about significant analgesic effect,which suggested that could exert importantfunction in the development/maintenance of bone cancer pain.
3.The shRNA lentivirus vectors based on PKCγgene sequence were constructedsuccessfully and lentivirus pLV-PKC_2 could specifically,highly efficiently inhibitPKCγgene expression.
4.When lentivirus pLV-PKC_2 was intrathecally injected to CIBP rats,it could generateevident analgesic effect and the expression of PKCγwas significantly down-regulated,which suggested that gene analgesic method mediated by siRNA could obtain definite,effective analgesic effect.
Major Conclusion:
In the development and maintenance of bone cancer pain,PKCγas an importantsignal molecular participates the transmission of nociceptive signal.After the lentiviruspLV-PKC_2 was intrathecally injected,it could bring about significant analgesic effect.
Innovation:
This study is a novel gene therapy based on the mechanism of CIBP.One side,itidentified signal molecular PKCγexerted important functions in the signal transmission ofCIBP;on the other hand,the lentivirus pLV-PKC_2 based on siRNA technology could produce significant analgesic effect,which suggested that gene analgesic therapy methodcould obtain definite,effective analgesic effect,and it could provide theory basis andexperiment basis to chronic pain therapy and the in vivo application of siRNA.
引文
1. Coleman RE. Clinical features of metastatic bone disease and risk of skeletal morbidity. Clin Cancer Res, 2006,12:6243s-6249s.
2.Sebastiano M, Fabio F. Management of painful bone metastases. Curr Opin Oncol,2007,19:308-314.
3. Thurlimann B, Toutz ND. Causes and treatment of bone pain of malignant origin[J].Drugs, 1996, 51:383-398.
4.Mundy GR. Metastasis to bone: causes, consequences and therapeutic opportunities [J]. Cancer, 2002, 2(8):584-593.
5. Goblirsch MJ, Zwolak PP, Clohisy DR. Biology of bone cancer pain. Clin Cancer Res 2006,12:6231s-6235s.
6. Coleman RE. Skeletal complications of malignancy. Cancer, 1997, 80:1588- 1594.
7. Portenoy RK, Lesage P. Management of cancer pain. Lancet, 1999, 353 ( 9165) : 1695.
8. Gordon-Williams RM, Dickenson AH. Central neuronal mechanisms in cancer-induced bone pain. Curr Opin Support Palliat Care, 2007, 1(1):6-10.
9. Mach DB, Rogers SD, Sabino MC, et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience, 2002,113: 155-166.
10. Mantyh PW, Clohisy DR, Koltzenburg M, et al. Molecular mechanisms of cancer pain. Cancer, 2002, 2:201 -209.
11. Martin WJ, Malmberg AB, Basbaum AI. PKC gamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain. Neurosci, 2001, 21:5321-5327.
12. Sweitzer SM, Wong SM, Peters MC, et al. Protein kinase C epsilon and gamma: involvement in formalin-induced nociception in neonatal rats. Pharmacol Exp Ther,2004, 309(2): 616-625.
13. Wen ZH, Guo YW, Chang YC, et al. D-2-amino-5-phosphonopentanoic acid inhibits intrathecal pertussis toxininduced thermal hyperalgesia and protein kinase C gamma up upregulation. Brain Res, 2003, 963(1-2): 1-7.
14. Mao-Ying QL, Zhao J, Dong ZQ, et al. A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. Biochem Biophys Res Commun, 2006, 345(4): 1292-1298.
15. Gopalkrishnan P, Sluka KA. Effect of varying frequency, intensity and pulse duration of TENS on primary hyperalgesia in inflamed rats [J] . Arch Phys Med Rehabil, 2000, 81:984-990.
16. Kathleen A, Sluka KA. Blockade of calcium channels can prevent the onset of secondary hyperalgesiaand allodynia induced by intradermal injection of capsaicin in rats [J] . Pain, 1997, 71:157-164.
17. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain, 2000, 87(2):149-158.
18. Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain, 2002,96(1-2):129-40.
19. Bhagat K, Chinyanga HM. Trends in cancer pain management. Cent Afr Med, 2000, 46(2):46-54.
20. Slatkin N. Cancer-related pain and its pharmacologic management in the patient with bone metastasis. Support Oncol, 2006, 4 (Suppl 1):15-21.
21. O'Toole GC, Boland P. Metastatic bone cancer pain: etiology and treatment options. Curr Pain Headache Rep, 2006,10:288-292.
22. Donovan-Rodriguez T, Dickenson AH, Urch CE. Gabapentin normalizes spinal neuronal responses that correlate with behavior in a rat model of cancer-induced bone pain. Anesthesiology, 2005,102(1):132-140.
23. Ringe JD, Body JJ. A review of bone pain relief with ibandronate and other bisphosphonates in disorders of increased bone turnover.Clin Exp Rheumatol, 2007,25(5):766-74.
24. Colvin L, Fallon M. Challenges in cancer pain management--bone pain. Eur Cancer, 2008, 44(8):1083-1090.
25. Preston SJ, Clifton-Bligh P, Laurent MR, et al. Effect of methotrexate and sulphasalazine on UMR 106 rat osteosarcoma cells. Br Rheumatol, 1997, 36 (2):178-184.
26. Honore P, Luger NM, Sabino MA, et al. Osteoprotegerin blocks bone cancer- induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord. Nat Med, 2000, 6(5): 521-528.
27. Gralow J, Tripathy D. Managing metastatic bone pain: the role of bisphosphonates. Pain Symptom Manage, 2007, 33(4):462-472.
28. Lussier D, Huskey AG, Portenoy RK. Adjuvant analgesics in cancer pain management. Oncologist, 2004, 9(5):571-591.
29. Terrier G, Ranoux D, Bourzeix JV, et al. Continuous nerve blocks in the management of bone pain due to compression by cancer metastasis: place in palliative care units. Palliat Care, 2008, 24(3): 190-191.
30. Cherny NJ, Chang V, Frager G, et al. Opioid pharmacotherapy in the management of cancer pain: a survey of strategies used by pain physicians for the selection of analgesic drugs and routes of administration. Cancer, 1995, 76(7):1283-1293.
31. Polgar E, Fowler JH, McGill MM, et al. The types of neuron which contain protein kinase C gamma in rat spinal cord. Brain Res. 1999; 833: 71-80.
32. Malmberg AB, Chen C, Tonigawa S, Basbaum AI. Preserved acute pain and reduced neuropathic pain in mice lacking PKCy [ J ] . Science, 1997, 278(4562):279-283.
33. Martin WJ, Liu H, Wang H, et al. Inflammation-induced up-regulation of protein kinase C_γ immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing [J]. Neuroscience, 1999, 88(4): 1267-1274.
34. Marie H, Petter V, Emelie S, et al. Inhibition of PKC activity blocks the increase of ETB receptor expression in cerebral arteries. BMC Pharmacology 2006, 6:13.
1.Martin WJ, Malmberg AB, Basbaum AI. PKC gamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain [J] . Neurosci, 2001, 21:5321-5327.
2.Sweitzer SM, Wong SM, Peters MC, et al. Protein kinase C epsilon and gamma:involvement in formalin-induced nociception in neonatal rats [J] . J Pharmacol Exp Ther, 2004, 309(2): 616-625.
3.Wen ZH, Guo YW, Chang YC, et al. D-2-amino-5-phosphonopentanoic acid inhibits intrathecal pertussis toxininduced thermal hyperalgesia and protein kinase C gamma up upregulation [J] . Brain Res, 2003, 963(1-2): 1-7.
4.Mao-Ying QL, Zhao J, Dong ZQ, et al. A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells[J]. Biochem Biophys Res Commun, 2006, 345(4): 1292-1298.
5.Sluka KA, Audette KM. Activation of protein kinase C in the spinal cord produces mechanical hyperalgesia by activating glutamate receptors, but does not mediate chronic muscle-induced hyperalgesia [J] . Molecular Pain, 2006, 2(13): 1-9.
6.Gopalkrishnan P, Sluka KA. Effect of varying frequency, intensity and pulse duration of TENS on primary hyperalgesia in inflamed rats [J] . Arch Phys Med Rehabil, 2000, 81:984-990.
7.Kathleen A, Sluka KA. Blockade of calcium channels can prevent the onset of secondary hyperalgesiaand allodynia induced by intradermal injection of capsaicin in rats [J] . Pain, 1997,71:157-164.
8.Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain, 2000, 87(2): 149-58.
9.Hua XY, Chen P, Yaksh TL. Inhibition of spinal protein kinase C reduces nerve injury-induced tactile allodynia in neuropathic rats. Neurosci Lett, 1999, 276: 99- 102.
10. Martin WJ, Liu H, Wang H, et al. Inflammation-induced up-regulation of protein kinase C_γ immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing [J] . Neuroscience, 1999, 88(4): 1267-1274.
11. Nishizuka Y. The molecular heterogeneity of protein kinase C and implacations for cellular regulation [J] . Nature, 1988, 334:661-665.
12. Toullec D, Pianetti P, Coste H, et al. The bisindolylmaleimide GF109203X is a potent and selective inhibitor of protein kinase C [J] .Biol Chem, 1991, 266:15771-15781.
13. Turan NN, Akar F, Budak B, et al. How DMSO, a widely used solvent, affects spinal cord injury [J] . Ann Vase Surg, 2008, 22(1): 98-105.
14. Colucci M, Maione F, Bonito MC, et al. New insights of dimethyl sulphoxide effects (DMSO) on experimental in vivo models of nociception and inflammation [J] . Pharmacol Res, 2008, 57(6):419-425.
15. Malmberg AB, Chen C, Tonigawa S, Basbaum AI. Preserved acute pain and reduced neuropathic pain in mice lacking PKC_γ[J] . Science, 1997, 278(4562):279-283.
1.Polgar E, Fowler JH, McGill MM, et al. The types of neuron which contain protein kinase C gamma in rat spinal cord. Brain Res. 1999, 833: 71-80.
2.Tannaka C, Saito N. Localization of subspecies of protein kinase C in the mammalian central nervous system. Neurochem Int. 1992, 21: 499-512.
3.Yu Fu, Jeong Han, Titilope Ishola, et al. PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior. Molecular Pain, 2008, 4:26.
4. Gonzalez-Alegre P. Therapeutic RNA interference for neurodegenerative diseases. Rev Neurol,2008,47(12):641-7.
5.Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296(5567): 550-553.
6.Schaffert D, Wagner E. Gene therapy progress and prospects: synthetic polymer- based systems. Gene Ther, 2008,15(16):1131-8.
7.Sweitzer SM, Wong SM, Peters MC, et al. Protein kinase C epsilon and gamma: involvement in formalin-induced nociception in neonatal rats. Pharmacol Exp Ther, 2004, 309(2): 616-625.
8.Martin WJ, Malmberg AB, Basbaum AI. PKC gamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain. Neurosci, 2001, 21:5321 -5327.
9. Malmberg AB, Chen C, Tonigawa S, et al. Preserved acute pain and reduced neuropathic pain in mice lacking PKC_γ [J].Science, 1997, 278(4562):279-283.
10.DeVincenzo J, Cehelsky JE, Alvarez R, et a1. Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res, 2008, 77(3):225-231.
11. Nokes JD, Cane PA. New strategies for control of respiratory syncytial virus infection. Curr Opin Infect Dis, 2008, 21(6):639-643.
12. Bernstein E, Caudy A, Hammond S M,et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J].Nature, 2001, 409:363-366.
13. Barbara AK, Kriton KMT, Peter TR. Inhibition of telomerase activity in human cancer cells by RNA interference. Mol Cancer Ther, 2003, 2:209.
14. Abbas-Terki T, Blanco-Bose W, Deglon N. Lentiviral-mediated RNA interference. Hum Gene Ther, 2002,13(18): 2197-2201.
15. Braun A. Current protocols in human genetics. United States , John Wiley, 2006, Chapter 12:Unit 12.1.
16. Rubinson D A, Dillon C P, Kwiatkowski A V, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003, 33: 401-406.
17. Spirin PV, Vil'gelm AE, Prasolov VS. Lentiviral vectors.Mol Biol, 2008,42(5):913-26.
18. Hommel J D, Sears R M, Georgescu D, et al. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med, 2003, 9:1539-1544.
19. Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference. NatBiotechnol, 2004, 22:326-330.
20. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(6836): 494-498.
1.Gopalkrishnan P, Sluka KA. Effect of varying frequency, intensity and pulse duration of TENS on primary hyperalgesia in inflamed rats [J] . Arch Phys Med Rehabil, 2000,81:984-990.
2.Kathleen A, Sluka KA. Blockade of calcium channels can prevent the onset of secondary hyperalgesiaand allodynia induced by intradermal injection of capsaicin in rats [J] . Pain, 1997,71:157-164.
3.Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain, 2000, 87(2):149-158.
4.Kanis JA, McCloskey EV. Bone turnover and biochemical markers in malignancy.Cancer, 1997, 80:1538-1545.
5.Mach DB, Rogers SD, Sabino MC, et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience, 2002, 113: 155- 166.
6.Mantyh PW, Clohisy DR, Koltzenburg M, et al. Molecular mechanisms of cancer pain. Nat Rev, Cancer 2002, 2:201-209.
7. Fairchild A, Chow E. Role of radiation therapy and radiopharmaceuticals in bone metastases.Curr Opin Support Palliat Care, 2007,1(3):169-173.
8.Nelson CJ, Lee JS, Gamboa MC, et al. Cognitive effects of hormone therapy in men with prostate cancer. Cancer, 2008,113(5):1097-1106.
9.de Leon-Casasola OA. Current developments in opioid therapy for management of cancer pain.Clin J Pain, 2008,10:S3-7.
10.Rodriguez-Menendez V, Tremolizzo L, Cavaletti G. Targeting cancer and neuropathy with histone deacetylase inhibitors: two birds with one stone. Curr Cancer Drug Targets,2008, 8(4):266-274.
11.Matzke M, Matzke AJ, Kooter JM. RNA: guiding gene silencing. Science, 2001, 293(5532):1080-1083.
12.Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411 (6836): 494-498.
13. Naito Y, Nohtomi K, Onogi T, et al. Optimal design and validation of antiviral siRNA for targeting HIV-1. Retrovirology, 2007, 4:80.
14. Ramaswamy S. Rational design of cancer-drug combinations.N Engl J Med, 2007,357(3):299-300.
15. Amarzguioui M, Lundberg P, Cantin E, et al. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat Protoc, 2006, 1(2):508-517.
16. Devi GR. siRNA-based approaches in cancer therapy. Cancer Gene Ther, 2006,13(9):8l9-829.
17. Gr(?)nweller A, Hartmann RK. RNA interference as a gene-specific approach for molecular medicine. Curr Med Chem, 2005,12(26):3143-3161.
18. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296(5567): 550-553.
19. Bernstein E, Caudy A, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J]. Nature, 2001, 409:363-366.
20. DeVincenzo J, Cehelsky JE, Alvarez R, et al. Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res, 2008, 77(3):225-231.
21. Nokes JD, Cane PA. New strategies for control of respiratory syncytial virus infection. Curr Opin Infect Dis, 2008, 21(6):639-643.
22. Abbas-Terki T, Blanco-Bose W, Deglon N. Lentiviral-mediated RNA interference. Hum Gene Ther, 2002,13(18): 2197-2201
23. Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003, 33: 401-406.
24. Hommel JD, Sears RM, Georgescu D, et al. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med, 3003, 9:1539-1544.
1.Feigner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient, lipid mediated DNAtransfection procedure [J]. Proc Natl Acad Sci, 1987, 84 (21): 7413-7417.
2.Audouy SA, Leij LF, Hoekstra D, et al. In vivo characteristics of cationic liposomes as delivery vectors for gene therapy[J]. Pharm Res, 2002,19(11): 1599-1605.
3.Shimizu K, Maitani Y, Takahashi N, et al. Association of liposomes containing a soybean-derived sterylglucoside mixture with raz primary cultured hepato- cytes [J].Biol Pharm Bull, 1998, 21:818-822.
4. Kawano K, Nakamura K, Hayashi K, et al. Liver targeting liposomes containing sitosterol glucoside with regard to penetration enhancing effect on HepG2 cells[J]. Pharm Bull, 2002, 25(6):766-770.
5. Guo W , Lee T, Sudimack J, el al. Receptor specific delivery of liposomes via folate PEG-Chol [J]. Liposome Res, 2000,10(2-3):179-195.
6.Song S, Liu D, Peng J, et al. Peptide ligand-mediated liposome distribution and targeting to EGFR expressing tumor in vivo.Int J Pharm, 2008, 363(1-2):155-161.
7. Sun X, Hai L, Wu Y, et al. Targeted gene delivery to hepatoma cells using galactosylated liposome-polycation-DNA complexes (LPD) [J]. DrugTargeting, 2005,13(2):121-128.
8. Chen Q, Tong S, Dewhirst MW, et al. Targeting tumor microvessels using doxorubicin encapsulated in a novel thermosensitive liposome [J]. Mol Cancer Ther, 2004,3(10):1311-1317.
9. Peer D, Margalit R, Tumor-targeted hyaluronan nanoliposomes increase the antitumor activity of liposomal doxorubicin in syngeneic and human xenograft mouse tumor models[J]. Neoplasia, 2004, 6(4):343-353.
10.Judge AD, Bola G, Lee AC, et al. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo[J]. Mol Ther, 2006,13(3): 494-505.
11. Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice[J]. Mol Biol, 2003,327(4): 761-766.
12. Yin C, Xi L, Wang X, et al. Silencing heat shock factor-1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia reperfusion injury in mice[J]. Mol Cell Cardiol, 2005, 39(4): 681-689.
13. Dora G, Patel S, Wotherspoon G, et al. siRNA relieves chronic neuropathic pain [J]. Nucleic Acids Res, 2004, 32(5): 223-228.
14. Thakker DR, Natt F, Husken D, et al. Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference [J]. Proc Natl Acad Sci , 2004, 101(49): 17270-17275.
15. Thakker DR, NattF, Husken D, et al. siRNA mediated knock down of the serotonin transporter in the adult mouse brain [J]. Mol Psychiatry, 2005, 10(8): 782-789.
16. Yoshizato K, Nishi T, Goto T, et al. Gene delivery with optimized electroporation parameters shows potential for treatment of gliomas [J]. Int Oncol, 2000, 16(5):899-905.
17. Wouters PC, Dutreux N, Smelt JPPM , et al. Effects of pulsed electric fields On inactivation kinetics of listeriainnoeua [J]. Appl Environ Microbiol, 1999, 65(12): 5364-5371.
18. Tyuin M, Padda R, Huang KX, et al. Electrotransformarion of clostridium acetobutylicum ATCC 824 using hight-votage radio frequency modulated square pulses[J]. Applied Microbiology, 2000, 88: 220-227.
19. Nanda GS, Mishra KP. Studies on electroprotion of thermally and chemically treated -human erythrocytes [J]. Bioelectrochem Bioenerge, 1994, 34: 129-134.
20. Cenlazar M, Miklavcic D, Scancar J, et al. Increased platirum accumulation in SA-1 tumour cells after in vivo electrochemotherapy with cisplatin. British Journal of Cancer, 1999, 79(9): 1386-1391.
21. Fujimoto H, Kato K, Iwata H. Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells. Anal Bioanal Chem, 2008, 392(7-8):1309-1316.
22. Yang WB, Hao F, Song ZQ, et al. Apoptosis of the dermal papilla cells of hair follicle associated with the expression of gene HSP-CO16 in vitro. Exp Dermatol, 2005,14(3):209-214.
23. Comes N, Borr(?)s T. Functional delivery of synthetic naked siRNA to the human trabecular meshwork in perfused organ cultures. Mol Vis, 2007,13:1363-74.
24. Jantsch J, Turza N, Volke M, et al. Small interfering RNA (siRNA) delivery into murine bone marrow-derived dendritic cells by electroporation[J]. Immunol Methods, 2008, 337(1):71-77.
25. Chun HJ, Zheng L, Ahmad M, et al. Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency. Nature, 2002,419(6905):395-399.
26. Han SY, Gai W, Yancovitz M, et al. Nucleofection is a highly effective gene transfer technique for human melanoma cell lines. Exp Dermatol, 2008, 17(5):405-11.
27. Gartner A, Collin L, Lalli G. Nucleofection of primary neurons. Methods Enzymol, 2006, 406:374-388.
28. Takahashi Y, Nishikawa M, Kobayashi N, et al. Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases [J]. Control Release,2005,105(3):332-343.
29. Schiffelers RM, Xu J, Storm G, et al. Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis. Arthritis Rheum, 2005,52(4):1314-1318,
30. Calegari F, Marzesco AM, Kittler R, et al. Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture. Differentiation, 2004,72(2-3): 92-102.
31. McMahon JM, Wells DJ. Electroporation for gene transfer to skeletal muscle: current status. BioDrugs, 2004, 18(3): 155-165.
32. Glasspool-Malone J, Somiari S, Drabick JJ, et al. Efficient nonviral cutaneous transfection. MolTher, 2000, 2(2): 140-146.
33. Yamashita YI, Shimada M, Hasegawa H, et al. Electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model [J]. Cancer Res, 2001,61(3): 1005-1012.
34. Dean DA, Machado-Aranda D, Blair-Parks K, et al. Electopo ration as a method for high-level nonviral gene transfer to the lung. GeIle Ther, 2003, 10(18): 1608-1615.
35. Rols MP, Delteil C, Golzio M, et al. In vivo electrically mediated protein and gene transfer in murine melanoma . Nat Biotechnol, 1998,16(2): 168-171.
36. Widera G, Austin M, Rabussay D, et al. Increased DNA vaccine delivery and immunogenicity by electroporation in vivo[J]. Imunol, 2000,164(9): 4635-4640.
37. Medi BM, Hoselton S, Marepalli RB, et al. Skin targeted DNA vaccine delivery using electroporation in rabbits.Ⅰ: eficacy. Int J Pharm, 2005,294(1-2): 53-63.
38. Scheertinek JP, Karlis J, Tjelle TE, et al. In vivo electroporation improve immune responses to DNA vaccination in sheep. Vaccine, 2004, 22 (13-14): 1820-1825.
39. Ottea G, Schaefer M, Doe B, et al. Enhancement of DNA vaccine potency in rhesus macaque by electroporation. Vaccine, 2004, 22(19): 2489-2493.
40. Momose T, Tonegawa A, Takeuchi J, et al. Efficient targeting of gene expression in chick embryos by microelectroporation. Development, Growth and Differentiation,1999, 41: 335-334.
41. Frank L, David YL, David AZ, et al. effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation [J]. Cancer Res. 2001, 61:3281- 3284.
42. Daud AI, DeConti RC, Andrews S, et al. Phase Ⅰ trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. Clin Oncol, 2008, 26(36): 5896-5903.
43. Derossi D, Joliot AH, Chassaing G, et al. The third helix of the Antennapedia homeodomain translocates through biological membranes. Biol Chem, 1994, 269(14):10444-10450.
44. Brugidou J, Legrand C, Mery J, et al. The retro-inverso form of a homeobox- derived short peptide is rapidly internalised by cultured neurones: a new basis for an efficient intracellular delivery system. Biochem Biophys Res Commun, 1995, 214(2):685- 693.
45. Williams EJ, Dunican DJ, Green PJ, et al. Selective inhibition of growth factor- stimulated mitogenesis by a cell-permeable Grb2-binding peptide. Biol Chem, 1997,272(35):22349-22354.
46. Wender PA, Jessop TC, Pattabiraman K, et al. An efficient, scalable synthesis of the molecular transporter octaarginine via a segment doubling strategy. Org Lett, 2001,3(21):3229-3232.
47. Pooga M, Kut C, Kihlmark M, et al. Cellular translocation of proteins by transportan.FASEB, 2001, 15(8):1451-1453.
48. Lindgren M, Gallet X, Soomets U, et al. Translocation properties of novel cell penetrating transportan and penetratin analogues. Bioconjug Chem, 2000, 11(5):619-26.
49. Derossi D, Chassaing G, Prochiantz A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol, 1998, 8(2):84-87.
50. Prochiantz A. Getting hydrophilic compounds into cells: lessons from homeo- peptides. Curr Opin Neurobiol, 1996, 6(5):629-634.
51. Wunderbaldinger P, Josephson L, Weissleder R. Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles. Bioconjug Chem, 2002,13(2):264-8.
52. Moschos SA, Jones SW, Perry MM, et al. Lung delivery studies using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjug Chem, 2007, 18(5): 1450- 1459.
53. Christiaens B, Symoens S, Verheyden S, et al. Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes. Eur J Biochem, 2002,269(12):2918-2926.
54. Gratton JP, Yu J, Griffith JW, et al. Vascular endothelial growth factor- dependent down-regulation of Flk-1/KDR involves Cbl-mediated ubiquitina- tion Consequences on nitric oxide production from endothelial cells[J]. Biol Chem, 2003, 278(22): 1-7.
55. Schwarze SR, Dowdy SF. In vivo protein transduction: Intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol Sci, 2000,21:45-48.
56. Turner JJ, Jones S, Fabani MM, et al. RNA targeting with peptide conjugates of oligonucleotides, siRNA and PNA. Blood Cells Mol Dis, 2007, 38(1): 1-7.
57. Moschos SA, Jones SW, Perry MM, et al. Lung delivery studies using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjug Chem, 2007, 18(5):1450-1459.
58. Brummelkarnp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002, 296(5567):550-553.
59. Diallo M, Arenz C, Schmitz K, et al. Long endogenous dsRNAs can induce complete gene silencing in mammalian cells and primary cultures. Oligonucleotides, 2003,13(5):381-392.
60. Xia XG, Zhou H, Ding H, et al. An enhanced U6 promoter for synthesis of short hairpin RNA. Nucleic Acids Res, 31(17):el00.
61. Miyagishi M, Taira K. Development and application of siRNA expression vector.Nucleic Acids Res Suppl, 2002, (2):113-114.
62. Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 2002,16(8):948-958.
63. Sui G, Soohoo C, Affar B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci, 99(8):5515-5520.
64. Vorburger SA, Hunt KK. Adenoviral gene therapy. Oneologist, 2002, 7(1): 46-59.
65. Kondo S, Terashima M, Fukuda H, et al. Gene engineering of the adenovirus vector.Uirusu, 2007, 57(1):37-45.
66. Chen LM, Le HY, Qin RY, et al. Reversal of the phenotype by K-rasval 12 silencing mediated by adenovirus delivered siRNA in human pancreatic cancer cell line Panc-1. World J Gastroenterol, 2005, 11(6):831-838.
67. Chetty C, Bhoopathi P, Joseph P, et al. Adenovirus mediated small interfeting RNA against matrix metalloproteinase-2 suppresses tumor growth and lung metastasis in mice. Mol Cancer Ther, 2006, 5(9):2289-2299.
68. Fukumoto A, Tomod a K, Yoneda-Kato N, et al. Depletion of Jab-1 inhibits proliferation of pancreatic cancer cell lines. FEBS Lett, 2006, 580(25):5836 -5844.
69. Naldini L. Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin Biotechnol, 1998, 9(5):457-463.
70. Lois C, Hong EJ, Pease S, et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science, 295(5556): 868-872.
71. Pfeifer A, Ikawa M, Dayn Y, et al. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci, 2002, 99(4):2140-2145.
72. Scherr M, Eder M. Gene transfer into hematopoietic stem cells using lentiviral vectors.Curr Gene Ther, 2(1):45-55.
73. Naldini L, Verma IM. Lentiviral vectors. Adv Virus Res, 2000, 55:599-609.
74. McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 21(6):639-44.
75. Szulc J, Wiznerowicz M, Sauvain MO, et al. A versatile tool for conditional gene expression and knockdown. Nat Methods, 2006, 3(2):109-16.
76. Johan J, Cecilia L. Lentiviral Vectors for Use in the Central Nervous System.Molecular Therapy, 2006,13: 484-493.
77. Wiznerowicz M, Trono D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. Virol, 2003, 77(16):8957-8961
78. Zhang L, Liu HJ, Li TJ, et al. Lentiviral vector-mediated siRNA knockdown of SR-PSOX inhibits foam cell formation in vitrol. Acta Pharmacol Sin, 2008, 29 (7):847-852.
79. Herold MJ, Vanden Brandt J, Seibler J, et al. Inducible and reversible gene silencing by stable integration of a shRNA-encoding lentivirus in transgenic rats1. Proc Natl Acad Sci, 2008, 105(47):18507-18512.
80. Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003, 33:401-406.
81. Lian SS, Ashley SW, Whang EE. Lentivirus-mediated RNA interference of HMGA1 promotes chemosensitivity to gemcitabine in pancreatic adeno-carcinoma. Gastrointest Surg, 2006,10:1254-1263
82. Lee SH, Choi SH, Kim SH, et al. Thermally sensitive cationic polymer nanocapsules for specific cytosolic delivery and efficient gene silencing of siRNA: swelling induced physical disruption of endosome by cold shock [J]. Control Release, 2008,125(1):25-32.
83. Kim JS, Yoon TJ, Yu KN. et al. Cellular uptake of magnetic nanoparticle is mediated through energy-dependent endocytosis in A549 cells[J]. Vet Sci, 2006, 7(4):321-326.
84. Fishbein I, Chomy M, Rabinovich L, et a1. Nanoparticulate delivery system of atyrphostin for the treatment of restenosis [J]. CotroI Rel, 2000, 65: 221-229.
85. Li W, Szoka FC. Lipid-based nanoparticles for nucleic acid delivery. Pharm Res, 2007,24(3): 438-449.
86. Akhtar S, Benter I. Toxicogenomics of nonviral drug delivery systems for RNAi: potential impact on siRNA-mediated gene silencing activity and specificity. Adv Drug Deliv, 2007, 59(2):1641-1682.
87. Lu PY, Xie F, Woodle MC. In vivo application of RNA interference: from functional genomics to therapeutics. Adv Genet, 2005, 54:117-142
88. Lee SH, Kim SH, Park TG. Intracellular siRNA delivery system using polyelectrolyte complex micelles prepared from VEGF siRNA-PEG conjugate and cationic fusogenic peptide. Biochem Biophys Res Commun, 2007, 357(2):511-516.
89. Dobson J. Gene therapy progress and prospects magnetic nanoparticle-based gene delivery [J]. Gene Ther, 2006,13(4):283-287.
90. Toub N, Bertrand JR, Tamaddon AM, et al. Efficacy of siRNA nanocapsules targeted against the EWS-Fli1 oncogene in Ewing sarcoma. Pharm Res, 2006, 23(5):892-900.
91. Bouclier C, Moine L, Hillaireau H, et al. Physicochemical characteristics and preliminary in vivo biological evaluation of nanocapsules loaded with siRNA targeting estrogen receptor alpha. Biomacromolecules, 2008, 9(10):2881-2890.
92. Gryziewicz L. Regulatory aspects of drug approval for macular degeneration. Adv Drug Deliv, 2005, 57(14):2092-2098.
93. DeVincenzo J, Cehelsky JE, Alvarez R, et al. Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res, 2008, 77(3):225-231.
94. Nokes JD, Cane PA. New strategies for control of respiratory syncytial virus infection. Curr Opin Infect Dis, 2008, 21(6):639-643.
2.Sebastiano M, Fabio F. Management of painful bone metastases. Curr Opin Oncol,2007,19:308-314.
3. Thurlimann B, Toutz ND. Causes and treatment of bone pain of malignant origin[J].Drugs, 1996, 51:383-398.
4.Mundy GR. Metastasis to bone: causes, consequences and therapeutic opportunities [J]. Cancer, 2002, 2(8):584-593.
5. Goblirsch MJ, Zwolak PP, Clohisy DR. Biology of bone cancer pain. Clin Cancer Res 2006,12:6231s-6235s.
6. Coleman RE. Skeletal complications of malignancy. Cancer, 1997, 80:1588- 1594.
7. Portenoy RK, Lesage P. Management of cancer pain. Lancet, 1999, 353 ( 9165) : 1695.
8. Gordon-Williams RM, Dickenson AH. Central neuronal mechanisms in cancer-induced bone pain. Curr Opin Support Palliat Care, 2007, 1(1):6-10.
9. Mach DB, Rogers SD, Sabino MC, et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience, 2002,113: 155-166.
10. Mantyh PW, Clohisy DR, Koltzenburg M, et al. Molecular mechanisms of cancer pain. Cancer, 2002, 2:201 -209.
11. Martin WJ, Malmberg AB, Basbaum AI. PKC gamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain. Neurosci, 2001, 21:5321-5327.
12. Sweitzer SM, Wong SM, Peters MC, et al. Protein kinase C epsilon and gamma: involvement in formalin-induced nociception in neonatal rats. Pharmacol Exp Ther,2004, 309(2): 616-625.
13. Wen ZH, Guo YW, Chang YC, et al. D-2-amino-5-phosphonopentanoic acid inhibits intrathecal pertussis toxininduced thermal hyperalgesia and protein kinase C gamma up upregulation. Brain Res, 2003, 963(1-2): 1-7.
14. Mao-Ying QL, Zhao J, Dong ZQ, et al. A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. Biochem Biophys Res Commun, 2006, 345(4): 1292-1298.
15. Gopalkrishnan P, Sluka KA. Effect of varying frequency, intensity and pulse duration of TENS on primary hyperalgesia in inflamed rats [J] . Arch Phys Med Rehabil, 2000, 81:984-990.
16. Kathleen A, Sluka KA. Blockade of calcium channels can prevent the onset of secondary hyperalgesiaand allodynia induced by intradermal injection of capsaicin in rats [J] . Pain, 1997, 71:157-164.
17. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain, 2000, 87(2):149-158.
18. Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain, 2002,96(1-2):129-40.
19. Bhagat K, Chinyanga HM. Trends in cancer pain management. Cent Afr Med, 2000, 46(2):46-54.
20. Slatkin N. Cancer-related pain and its pharmacologic management in the patient with bone metastasis. Support Oncol, 2006, 4 (Suppl 1):15-21.
21. O'Toole GC, Boland P. Metastatic bone cancer pain: etiology and treatment options. Curr Pain Headache Rep, 2006,10:288-292.
22. Donovan-Rodriguez T, Dickenson AH, Urch CE. Gabapentin normalizes spinal neuronal responses that correlate with behavior in a rat model of cancer-induced bone pain. Anesthesiology, 2005,102(1):132-140.
23. Ringe JD, Body JJ. A review of bone pain relief with ibandronate and other bisphosphonates in disorders of increased bone turnover.Clin Exp Rheumatol, 2007,25(5):766-74.
24. Colvin L, Fallon M. Challenges in cancer pain management--bone pain. Eur Cancer, 2008, 44(8):1083-1090.
25. Preston SJ, Clifton-Bligh P, Laurent MR, et al. Effect of methotrexate and sulphasalazine on UMR 106 rat osteosarcoma cells. Br Rheumatol, 1997, 36 (2):178-184.
26. Honore P, Luger NM, Sabino MA, et al. Osteoprotegerin blocks bone cancer- induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord. Nat Med, 2000, 6(5): 521-528.
27. Gralow J, Tripathy D. Managing metastatic bone pain: the role of bisphosphonates. Pain Symptom Manage, 2007, 33(4):462-472.
28. Lussier D, Huskey AG, Portenoy RK. Adjuvant analgesics in cancer pain management. Oncologist, 2004, 9(5):571-591.
29. Terrier G, Ranoux D, Bourzeix JV, et al. Continuous nerve blocks in the management of bone pain due to compression by cancer metastasis: place in palliative care units. Palliat Care, 2008, 24(3): 190-191.
30. Cherny NJ, Chang V, Frager G, et al. Opioid pharmacotherapy in the management of cancer pain: a survey of strategies used by pain physicians for the selection of analgesic drugs and routes of administration. Cancer, 1995, 76(7):1283-1293.
31. Polgar E, Fowler JH, McGill MM, et al. The types of neuron which contain protein kinase C gamma in rat spinal cord. Brain Res. 1999; 833: 71-80.
32. Malmberg AB, Chen C, Tonigawa S, Basbaum AI. Preserved acute pain and reduced neuropathic pain in mice lacking PKCy [ J ] . Science, 1997, 278(4562):279-283.
33. Martin WJ, Liu H, Wang H, et al. Inflammation-induced up-regulation of protein kinase C_γ immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing [J]. Neuroscience, 1999, 88(4): 1267-1274.
34. Marie H, Petter V, Emelie S, et al. Inhibition of PKC activity blocks the increase of ETB receptor expression in cerebral arteries. BMC Pharmacology 2006, 6:13.
1.Martin WJ, Malmberg AB, Basbaum AI. PKC gamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain [J] . Neurosci, 2001, 21:5321-5327.
2.Sweitzer SM, Wong SM, Peters MC, et al. Protein kinase C epsilon and gamma:involvement in formalin-induced nociception in neonatal rats [J] . J Pharmacol Exp Ther, 2004, 309(2): 616-625.
3.Wen ZH, Guo YW, Chang YC, et al. D-2-amino-5-phosphonopentanoic acid inhibits intrathecal pertussis toxininduced thermal hyperalgesia and protein kinase C gamma up upregulation [J] . Brain Res, 2003, 963(1-2): 1-7.
4.Mao-Ying QL, Zhao J, Dong ZQ, et al. A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells[J]. Biochem Biophys Res Commun, 2006, 345(4): 1292-1298.
5.Sluka KA, Audette KM. Activation of protein kinase C in the spinal cord produces mechanical hyperalgesia by activating glutamate receptors, but does not mediate chronic muscle-induced hyperalgesia [J] . Molecular Pain, 2006, 2(13): 1-9.
6.Gopalkrishnan P, Sluka KA. Effect of varying frequency, intensity and pulse duration of TENS on primary hyperalgesia in inflamed rats [J] . Arch Phys Med Rehabil, 2000, 81:984-990.
7.Kathleen A, Sluka KA. Blockade of calcium channels can prevent the onset of secondary hyperalgesiaand allodynia induced by intradermal injection of capsaicin in rats [J] . Pain, 1997,71:157-164.
8.Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain, 2000, 87(2): 149-58.
9.Hua XY, Chen P, Yaksh TL. Inhibition of spinal protein kinase C reduces nerve injury-induced tactile allodynia in neuropathic rats. Neurosci Lett, 1999, 276: 99- 102.
10. Martin WJ, Liu H, Wang H, et al. Inflammation-induced up-regulation of protein kinase C_γ immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing [J] . Neuroscience, 1999, 88(4): 1267-1274.
11. Nishizuka Y. The molecular heterogeneity of protein kinase C and implacations for cellular regulation [J] . Nature, 1988, 334:661-665.
12. Toullec D, Pianetti P, Coste H, et al. The bisindolylmaleimide GF109203X is a potent and selective inhibitor of protein kinase C [J] .Biol Chem, 1991, 266:15771-15781.
13. Turan NN, Akar F, Budak B, et al. How DMSO, a widely used solvent, affects spinal cord injury [J] . Ann Vase Surg, 2008, 22(1): 98-105.
14. Colucci M, Maione F, Bonito MC, et al. New insights of dimethyl sulphoxide effects (DMSO) on experimental in vivo models of nociception and inflammation [J] . Pharmacol Res, 2008, 57(6):419-425.
15. Malmberg AB, Chen C, Tonigawa S, Basbaum AI. Preserved acute pain and reduced neuropathic pain in mice lacking PKC_γ[J] . Science, 1997, 278(4562):279-283.
1.Polgar E, Fowler JH, McGill MM, et al. The types of neuron which contain protein kinase C gamma in rat spinal cord. Brain Res. 1999, 833: 71-80.
2.Tannaka C, Saito N. Localization of subspecies of protein kinase C in the mammalian central nervous system. Neurochem Int. 1992, 21: 499-512.
3.Yu Fu, Jeong Han, Titilope Ishola, et al. PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior. Molecular Pain, 2008, 4:26.
4. Gonzalez-Alegre P. Therapeutic RNA interference for neurodegenerative diseases. Rev Neurol,2008,47(12):641-7.
5.Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296(5567): 550-553.
6.Schaffert D, Wagner E. Gene therapy progress and prospects: synthetic polymer- based systems. Gene Ther, 2008,15(16):1131-8.
7.Sweitzer SM, Wong SM, Peters MC, et al. Protein kinase C epsilon and gamma: involvement in formalin-induced nociception in neonatal rats. Pharmacol Exp Ther, 2004, 309(2): 616-625.
8.Martin WJ, Malmberg AB, Basbaum AI. PKC gamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain. Neurosci, 2001, 21:5321 -5327.
9. Malmberg AB, Chen C, Tonigawa S, et al. Preserved acute pain and reduced neuropathic pain in mice lacking PKC_γ [J].Science, 1997, 278(4562):279-283.
10.DeVincenzo J, Cehelsky JE, Alvarez R, et a1. Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res, 2008, 77(3):225-231.
11. Nokes JD, Cane PA. New strategies for control of respiratory syncytial virus infection. Curr Opin Infect Dis, 2008, 21(6):639-643.
12. Bernstein E, Caudy A, Hammond S M,et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J].Nature, 2001, 409:363-366.
13. Barbara AK, Kriton KMT, Peter TR. Inhibition of telomerase activity in human cancer cells by RNA interference. Mol Cancer Ther, 2003, 2:209.
14. Abbas-Terki T, Blanco-Bose W, Deglon N. Lentiviral-mediated RNA interference. Hum Gene Ther, 2002,13(18): 2197-2201.
15. Braun A. Current protocols in human genetics. United States , John Wiley, 2006, Chapter 12:Unit 12.1.
16. Rubinson D A, Dillon C P, Kwiatkowski A V, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003, 33: 401-406.
17. Spirin PV, Vil'gelm AE, Prasolov VS. Lentiviral vectors.Mol Biol, 2008,42(5):913-26.
18. Hommel J D, Sears R M, Georgescu D, et al. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med, 2003, 9:1539-1544.
19. Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference. NatBiotechnol, 2004, 22:326-330.
20. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(6836): 494-498.
1.Gopalkrishnan P, Sluka KA. Effect of varying frequency, intensity and pulse duration of TENS on primary hyperalgesia in inflamed rats [J] . Arch Phys Med Rehabil, 2000,81:984-990.
2.Kathleen A, Sluka KA. Blockade of calcium channels can prevent the onset of secondary hyperalgesiaand allodynia induced by intradermal injection of capsaicin in rats [J] . Pain, 1997,71:157-164.
3.Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain, 2000, 87(2):149-158.
4.Kanis JA, McCloskey EV. Bone turnover and biochemical markers in malignancy.Cancer, 1997, 80:1538-1545.
5.Mach DB, Rogers SD, Sabino MC, et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience, 2002, 113: 155- 166.
6.Mantyh PW, Clohisy DR, Koltzenburg M, et al. Molecular mechanisms of cancer pain. Nat Rev, Cancer 2002, 2:201-209.
7. Fairchild A, Chow E. Role of radiation therapy and radiopharmaceuticals in bone metastases.Curr Opin Support Palliat Care, 2007,1(3):169-173.
8.Nelson CJ, Lee JS, Gamboa MC, et al. Cognitive effects of hormone therapy in men with prostate cancer. Cancer, 2008,113(5):1097-1106.
9.de Leon-Casasola OA. Current developments in opioid therapy for management of cancer pain.Clin J Pain, 2008,10:S3-7.
10.Rodriguez-Menendez V, Tremolizzo L, Cavaletti G. Targeting cancer and neuropathy with histone deacetylase inhibitors: two birds with one stone. Curr Cancer Drug Targets,2008, 8(4):266-274.
11.Matzke M, Matzke AJ, Kooter JM. RNA: guiding gene silencing. Science, 2001, 293(5532):1080-1083.
12.Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411 (6836): 494-498.
13. Naito Y, Nohtomi K, Onogi T, et al. Optimal design and validation of antiviral siRNA for targeting HIV-1. Retrovirology, 2007, 4:80.
14. Ramaswamy S. Rational design of cancer-drug combinations.N Engl J Med, 2007,357(3):299-300.
15. Amarzguioui M, Lundberg P, Cantin E, et al. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat Protoc, 2006, 1(2):508-517.
16. Devi GR. siRNA-based approaches in cancer therapy. Cancer Gene Ther, 2006,13(9):8l9-829.
17. Gr(?)nweller A, Hartmann RK. RNA interference as a gene-specific approach for molecular medicine. Curr Med Chem, 2005,12(26):3143-3161.
18. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296(5567): 550-553.
19. Bernstein E, Caudy A, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J]. Nature, 2001, 409:363-366.
20. DeVincenzo J, Cehelsky JE, Alvarez R, et al. Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res, 2008, 77(3):225-231.
21. Nokes JD, Cane PA. New strategies for control of respiratory syncytial virus infection. Curr Opin Infect Dis, 2008, 21(6):639-643.
22. Abbas-Terki T, Blanco-Bose W, Deglon N. Lentiviral-mediated RNA interference. Hum Gene Ther, 2002,13(18): 2197-2201
23. Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003, 33: 401-406.
24. Hommel JD, Sears RM, Georgescu D, et al. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med, 3003, 9:1539-1544.
1.Feigner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient, lipid mediated DNAtransfection procedure [J]. Proc Natl Acad Sci, 1987, 84 (21): 7413-7417.
2.Audouy SA, Leij LF, Hoekstra D, et al. In vivo characteristics of cationic liposomes as delivery vectors for gene therapy[J]. Pharm Res, 2002,19(11): 1599-1605.
3.Shimizu K, Maitani Y, Takahashi N, et al. Association of liposomes containing a soybean-derived sterylglucoside mixture with raz primary cultured hepato- cytes [J].Biol Pharm Bull, 1998, 21:818-822.
4. Kawano K, Nakamura K, Hayashi K, et al. Liver targeting liposomes containing sitosterol glucoside with regard to penetration enhancing effect on HepG2 cells[J]. Pharm Bull, 2002, 25(6):766-770.
5. Guo W , Lee T, Sudimack J, el al. Receptor specific delivery of liposomes via folate PEG-Chol [J]. Liposome Res, 2000,10(2-3):179-195.
6.Song S, Liu D, Peng J, et al. Peptide ligand-mediated liposome distribution and targeting to EGFR expressing tumor in vivo.Int J Pharm, 2008, 363(1-2):155-161.
7. Sun X, Hai L, Wu Y, et al. Targeted gene delivery to hepatoma cells using galactosylated liposome-polycation-DNA complexes (LPD) [J]. DrugTargeting, 2005,13(2):121-128.
8. Chen Q, Tong S, Dewhirst MW, et al. Targeting tumor microvessels using doxorubicin encapsulated in a novel thermosensitive liposome [J]. Mol Cancer Ther, 2004,3(10):1311-1317.
9. Peer D, Margalit R, Tumor-targeted hyaluronan nanoliposomes increase the antitumor activity of liposomal doxorubicin in syngeneic and human xenograft mouse tumor models[J]. Neoplasia, 2004, 6(4):343-353.
10.Judge AD, Bola G, Lee AC, et al. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo[J]. Mol Ther, 2006,13(3): 494-505.
11. Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice[J]. Mol Biol, 2003,327(4): 761-766.
12. Yin C, Xi L, Wang X, et al. Silencing heat shock factor-1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia reperfusion injury in mice[J]. Mol Cell Cardiol, 2005, 39(4): 681-689.
13. Dora G, Patel S, Wotherspoon G, et al. siRNA relieves chronic neuropathic pain [J]. Nucleic Acids Res, 2004, 32(5): 223-228.
14. Thakker DR, Natt F, Husken D, et al. Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference [J]. Proc Natl Acad Sci , 2004, 101(49): 17270-17275.
15. Thakker DR, NattF, Husken D, et al. siRNA mediated knock down of the serotonin transporter in the adult mouse brain [J]. Mol Psychiatry, 2005, 10(8): 782-789.
16. Yoshizato K, Nishi T, Goto T, et al. Gene delivery with optimized electroporation parameters shows potential for treatment of gliomas [J]. Int Oncol, 2000, 16(5):899-905.
17. Wouters PC, Dutreux N, Smelt JPPM , et al. Effects of pulsed electric fields On inactivation kinetics of listeriainnoeua [J]. Appl Environ Microbiol, 1999, 65(12): 5364-5371.
18. Tyuin M, Padda R, Huang KX, et al. Electrotransformarion of clostridium acetobutylicum ATCC 824 using hight-votage radio frequency modulated square pulses[J]. Applied Microbiology, 2000, 88: 220-227.
19. Nanda GS, Mishra KP. Studies on electroprotion of thermally and chemically treated -human erythrocytes [J]. Bioelectrochem Bioenerge, 1994, 34: 129-134.
20. Cenlazar M, Miklavcic D, Scancar J, et al. Increased platirum accumulation in SA-1 tumour cells after in vivo electrochemotherapy with cisplatin. British Journal of Cancer, 1999, 79(9): 1386-1391.
21. Fujimoto H, Kato K, Iwata H. Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells. Anal Bioanal Chem, 2008, 392(7-8):1309-1316.
22. Yang WB, Hao F, Song ZQ, et al. Apoptosis of the dermal papilla cells of hair follicle associated with the expression of gene HSP-CO16 in vitro. Exp Dermatol, 2005,14(3):209-214.
23. Comes N, Borr(?)s T. Functional delivery of synthetic naked siRNA to the human trabecular meshwork in perfused organ cultures. Mol Vis, 2007,13:1363-74.
24. Jantsch J, Turza N, Volke M, et al. Small interfering RNA (siRNA) delivery into murine bone marrow-derived dendritic cells by electroporation[J]. Immunol Methods, 2008, 337(1):71-77.
25. Chun HJ, Zheng L, Ahmad M, et al. Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency. Nature, 2002,419(6905):395-399.
26. Han SY, Gai W, Yancovitz M, et al. Nucleofection is a highly effective gene transfer technique for human melanoma cell lines. Exp Dermatol, 2008, 17(5):405-11.
27. Gartner A, Collin L, Lalli G. Nucleofection of primary neurons. Methods Enzymol, 2006, 406:374-388.
28. Takahashi Y, Nishikawa M, Kobayashi N, et al. Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases [J]. Control Release,2005,105(3):332-343.
29. Schiffelers RM, Xu J, Storm G, et al. Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis. Arthritis Rheum, 2005,52(4):1314-1318,
30. Calegari F, Marzesco AM, Kittler R, et al. Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture. Differentiation, 2004,72(2-3): 92-102.
31. McMahon JM, Wells DJ. Electroporation for gene transfer to skeletal muscle: current status. BioDrugs, 2004, 18(3): 155-165.
32. Glasspool-Malone J, Somiari S, Drabick JJ, et al. Efficient nonviral cutaneous transfection. MolTher, 2000, 2(2): 140-146.
33. Yamashita YI, Shimada M, Hasegawa H, et al. Electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model [J]. Cancer Res, 2001,61(3): 1005-1012.
34. Dean DA, Machado-Aranda D, Blair-Parks K, et al. Electopo ration as a method for high-level nonviral gene transfer to the lung. GeIle Ther, 2003, 10(18): 1608-1615.
35. Rols MP, Delteil C, Golzio M, et al. In vivo electrically mediated protein and gene transfer in murine melanoma . Nat Biotechnol, 1998,16(2): 168-171.
36. Widera G, Austin M, Rabussay D, et al. Increased DNA vaccine delivery and immunogenicity by electroporation in vivo[J]. Imunol, 2000,164(9): 4635-4640.
37. Medi BM, Hoselton S, Marepalli RB, et al. Skin targeted DNA vaccine delivery using electroporation in rabbits.Ⅰ: eficacy. Int J Pharm, 2005,294(1-2): 53-63.
38. Scheertinek JP, Karlis J, Tjelle TE, et al. In vivo electroporation improve immune responses to DNA vaccination in sheep. Vaccine, 2004, 22 (13-14): 1820-1825.
39. Ottea G, Schaefer M, Doe B, et al. Enhancement of DNA vaccine potency in rhesus macaque by electroporation. Vaccine, 2004, 22(19): 2489-2493.
40. Momose T, Tonegawa A, Takeuchi J, et al. Efficient targeting of gene expression in chick embryos by microelectroporation. Development, Growth and Differentiation,1999, 41: 335-334.
41. Frank L, David YL, David AZ, et al. effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation [J]. Cancer Res. 2001, 61:3281- 3284.
42. Daud AI, DeConti RC, Andrews S, et al. Phase Ⅰ trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. Clin Oncol, 2008, 26(36): 5896-5903.
43. Derossi D, Joliot AH, Chassaing G, et al. The third helix of the Antennapedia homeodomain translocates through biological membranes. Biol Chem, 1994, 269(14):10444-10450.
44. Brugidou J, Legrand C, Mery J, et al. The retro-inverso form of a homeobox- derived short peptide is rapidly internalised by cultured neurones: a new basis for an efficient intracellular delivery system. Biochem Biophys Res Commun, 1995, 214(2):685- 693.
45. Williams EJ, Dunican DJ, Green PJ, et al. Selective inhibition of growth factor- stimulated mitogenesis by a cell-permeable Grb2-binding peptide. Biol Chem, 1997,272(35):22349-22354.
46. Wender PA, Jessop TC, Pattabiraman K, et al. An efficient, scalable synthesis of the molecular transporter octaarginine via a segment doubling strategy. Org Lett, 2001,3(21):3229-3232.
47. Pooga M, Kut C, Kihlmark M, et al. Cellular translocation of proteins by transportan.FASEB, 2001, 15(8):1451-1453.
48. Lindgren M, Gallet X, Soomets U, et al. Translocation properties of novel cell penetrating transportan and penetratin analogues. Bioconjug Chem, 2000, 11(5):619-26.
49. Derossi D, Chassaing G, Prochiantz A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol, 1998, 8(2):84-87.
50. Prochiantz A. Getting hydrophilic compounds into cells: lessons from homeo- peptides. Curr Opin Neurobiol, 1996, 6(5):629-634.
51. Wunderbaldinger P, Josephson L, Weissleder R. Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles. Bioconjug Chem, 2002,13(2):264-8.
52. Moschos SA, Jones SW, Perry MM, et al. Lung delivery studies using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjug Chem, 2007, 18(5): 1450- 1459.
53. Christiaens B, Symoens S, Verheyden S, et al. Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes. Eur J Biochem, 2002,269(12):2918-2926.
54. Gratton JP, Yu J, Griffith JW, et al. Vascular endothelial growth factor- dependent down-regulation of Flk-1/KDR involves Cbl-mediated ubiquitina- tion Consequences on nitric oxide production from endothelial cells[J]. Biol Chem, 2003, 278(22): 1-7.
55. Schwarze SR, Dowdy SF. In vivo protein transduction: Intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol Sci, 2000,21:45-48.
56. Turner JJ, Jones S, Fabani MM, et al. RNA targeting with peptide conjugates of oligonucleotides, siRNA and PNA. Blood Cells Mol Dis, 2007, 38(1): 1-7.
57. Moschos SA, Jones SW, Perry MM, et al. Lung delivery studies using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjug Chem, 2007, 18(5):1450-1459.
58. Brummelkarnp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002, 296(5567):550-553.
59. Diallo M, Arenz C, Schmitz K, et al. Long endogenous dsRNAs can induce complete gene silencing in mammalian cells and primary cultures. Oligonucleotides, 2003,13(5):381-392.
60. Xia XG, Zhou H, Ding H, et al. An enhanced U6 promoter for synthesis of short hairpin RNA. Nucleic Acids Res, 31(17):el00.
61. Miyagishi M, Taira K. Development and application of siRNA expression vector.Nucleic Acids Res Suppl, 2002, (2):113-114.
62. Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 2002,16(8):948-958.
63. Sui G, Soohoo C, Affar B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci, 99(8):5515-5520.
64. Vorburger SA, Hunt KK. Adenoviral gene therapy. Oneologist, 2002, 7(1): 46-59.
65. Kondo S, Terashima M, Fukuda H, et al. Gene engineering of the adenovirus vector.Uirusu, 2007, 57(1):37-45.
66. Chen LM, Le HY, Qin RY, et al. Reversal of the phenotype by K-rasval 12 silencing mediated by adenovirus delivered siRNA in human pancreatic cancer cell line Panc-1. World J Gastroenterol, 2005, 11(6):831-838.
67. Chetty C, Bhoopathi P, Joseph P, et al. Adenovirus mediated small interfeting RNA against matrix metalloproteinase-2 suppresses tumor growth and lung metastasis in mice. Mol Cancer Ther, 2006, 5(9):2289-2299.
68. Fukumoto A, Tomod a K, Yoneda-Kato N, et al. Depletion of Jab-1 inhibits proliferation of pancreatic cancer cell lines. FEBS Lett, 2006, 580(25):5836 -5844.
69. Naldini L. Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin Biotechnol, 1998, 9(5):457-463.
70. Lois C, Hong EJ, Pease S, et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science, 295(5556): 868-872.
71. Pfeifer A, Ikawa M, Dayn Y, et al. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci, 2002, 99(4):2140-2145.
72. Scherr M, Eder M. Gene transfer into hematopoietic stem cells using lentiviral vectors.Curr Gene Ther, 2(1):45-55.
73. Naldini L, Verma IM. Lentiviral vectors. Adv Virus Res, 2000, 55:599-609.
74. McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 21(6):639-44.
75. Szulc J, Wiznerowicz M, Sauvain MO, et al. A versatile tool for conditional gene expression and knockdown. Nat Methods, 2006, 3(2):109-16.
76. Johan J, Cecilia L. Lentiviral Vectors for Use in the Central Nervous System.Molecular Therapy, 2006,13: 484-493.
77. Wiznerowicz M, Trono D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. Virol, 2003, 77(16):8957-8961
78. Zhang L, Liu HJ, Li TJ, et al. Lentiviral vector-mediated siRNA knockdown of SR-PSOX inhibits foam cell formation in vitrol. Acta Pharmacol Sin, 2008, 29 (7):847-852.
79. Herold MJ, Vanden Brandt J, Seibler J, et al. Inducible and reversible gene silencing by stable integration of a shRNA-encoding lentivirus in transgenic rats1. Proc Natl Acad Sci, 2008, 105(47):18507-18512.
80. Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003, 33:401-406.
81. Lian SS, Ashley SW, Whang EE. Lentivirus-mediated RNA interference of HMGA1 promotes chemosensitivity to gemcitabine in pancreatic adeno-carcinoma. Gastrointest Surg, 2006,10:1254-1263
82. Lee SH, Choi SH, Kim SH, et al. Thermally sensitive cationic polymer nanocapsules for specific cytosolic delivery and efficient gene silencing of siRNA: swelling induced physical disruption of endosome by cold shock [J]. Control Release, 2008,125(1):25-32.
83. Kim JS, Yoon TJ, Yu KN. et al. Cellular uptake of magnetic nanoparticle is mediated through energy-dependent endocytosis in A549 cells[J]. Vet Sci, 2006, 7(4):321-326.
84. Fishbein I, Chomy M, Rabinovich L, et a1. Nanoparticulate delivery system of atyrphostin for the treatment of restenosis [J]. CotroI Rel, 2000, 65: 221-229.
85. Li W, Szoka FC. Lipid-based nanoparticles for nucleic acid delivery. Pharm Res, 2007,24(3): 438-449.
86. Akhtar S, Benter I. Toxicogenomics of nonviral drug delivery systems for RNAi: potential impact on siRNA-mediated gene silencing activity and specificity. Adv Drug Deliv, 2007, 59(2):1641-1682.
87. Lu PY, Xie F, Woodle MC. In vivo application of RNA interference: from functional genomics to therapeutics. Adv Genet, 2005, 54:117-142
88. Lee SH, Kim SH, Park TG. Intracellular siRNA delivery system using polyelectrolyte complex micelles prepared from VEGF siRNA-PEG conjugate and cationic fusogenic peptide. Biochem Biophys Res Commun, 2007, 357(2):511-516.
89. Dobson J. Gene therapy progress and prospects magnetic nanoparticle-based gene delivery [J]. Gene Ther, 2006,13(4):283-287.
90. Toub N, Bertrand JR, Tamaddon AM, et al. Efficacy of siRNA nanocapsules targeted against the EWS-Fli1 oncogene in Ewing sarcoma. Pharm Res, 2006, 23(5):892-900.
91. Bouclier C, Moine L, Hillaireau H, et al. Physicochemical characteristics and preliminary in vivo biological evaluation of nanocapsules loaded with siRNA targeting estrogen receptor alpha. Biomacromolecules, 2008, 9(10):2881-2890.
92. Gryziewicz L. Regulatory aspects of drug approval for macular degeneration. Adv Drug Deliv, 2005, 57(14):2092-2098.
93. DeVincenzo J, Cehelsky JE, Alvarez R, et al. Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res, 2008, 77(3):225-231.
94. Nokes JD, Cane PA. New strategies for control of respiratory syncytial virus infection. Curr Opin Infect Dis, 2008, 21(6):639-643.