【作者】 肖兴鹏；

【导师】 田玉科；

【作者基本信息】 华中科技大学 ， 麻醉学， 2009， 博士

【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（4×10⁴ 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H₁启动子后获得重组质粒（pLV-PKC₁、pLV-PKC₂、pLV-PKC₃），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-PKC₂、pLV-PKC₃可明显抑制PKCγ蛋白的表达水平，其中以慢病毒pLV-PKC₂（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-PKC₂组（鞘内注入10μl慢病毒pLV-PKC₂）。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-PKC₂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-PKC₂组大鼠在鞘内注射慢病毒pLV-PKC₂ 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-PKC₂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-PKC₂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-PKC₂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-PKC₂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-PKC₂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。更多还原

【Abstract】 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 Results1:The expression and significance of PKCγin spinal dorsal horn in a ratmodel of cancer bone painMethods: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 painMethods: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 efficiencyMethods: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-PKC₂、pLV-PKC₃could evidently decrease the expression of PKCγ,especially the inhibition efficiency ofpLV-PKC₂ （target site:+1913-+1931）) was the highest.4:Effect of analgesia by intrathecal injection ofPKCγ-shRNA lentivirus ina rat model of bone cancer painMethods: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-PKC₂ group （intrathecal injection of lentiviruspLV-PKC₂ 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-PKC₂ group significantly increased and the MWD evidently prolonged compared toCIBP group after 5 days in intrathecal injection of lentivirns pLV-PKC₂ (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-PKC₂ group significantly reduced compared to CIBP group （P＜0.05）.5:Statistical analysisAll 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-PKC₂ could specifically,highly efficiently inhibitPKCγgene expression.4.When lentivirus pLV-PKC₂ 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-PKC₂ 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-PKC₂ 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.更多还原