【作者】 王维；

【导师】 冯泽国；

【作者基本信息】 中国人民解放军军医进修学院 ， 麻醉学， 2010， 硕士

【摘要】 N-型钙通道是治疗慢性神经痛的一个重要药理学靶点,目前临床上应用的N型钙通道阻断剂不仅影响伤害性感受传导通路的功能,也影响中枢神经系统及自主神经系统的功能。有研究发现N-型钙通道CaV2.2e37a亚型特异地表达于背根节神经元,本课题组在前期的研究中已经设计筛选出针对CaV2.2e37a有特异性抑制作用的siRNA,并在293FT细胞上验证了该siRNA可以特异性的抑制与其共转染的CaV2.2e37a全长基因的表达。本研究拟用慢病毒载体携带该siRNA治疗CCI大鼠神经痛,观察其镇痛效果及毒副作用,为进一步研发以CaV2.2e37a为靶点的镇痛药物提供实验依据。第一部分针对CaV2.2e37a基因的siRNA慢病毒载体的构建与鉴定目的：构建针对CaV2.2e37a基因的siRNA慢病毒载体,为研究抑制CaV2.2e37a基因表达治疗神经痛打下基础。方法：针对已经筛选确定的有效沉默CaV2.2e37a基因的siRNA序列,合成该序列的OligoDNA,经退火形成双链DNA,与经Hpa I和Xho I双酶切后的pLL3.7载体连接,构建pLL3.7-CaV2.2e37a干扰质粒,挑取重组阳性克隆行测序鉴定。pLL3.7含有能持续表达小RNA的元件,同时能表达绿色荧光蛋白(GFP),将pRsv-REV、pMD1g-pRRE、pMD2G、pLL3.7-CaV2.2e37a共转染293T细胞,荧光定量PCR测定病毒转导滴度。结果：测序结果表明合成的siRNA核苷酸序列正确插入到慢病毒载体中,说明干扰质粒构建成功。浓缩病毒悬液的滴度为1×109Tu／ml。结论：成功构建了针对CaV2.2e37a基因干扰序列的慢病毒载体LV-shCaV2.2e37a。第二部分CCI大鼠模型的建立及其背根神经节中CaV2.2表达的变化目的：建立大鼠慢性坐骨神经压迫模型,检测CCI大鼠DRG中CaV2.2表达的变化。方法：大鼠CCI模型的建立及行为学测定：健康雄性SD大鼠20只,随机分为2组(n=10),CCI组：切开大鼠右侧大腿皮肤,钝性分离肌肉,暴露右侧坐骨神经,4-0铬制肠线围绕坐骨神经干均匀打4个间隔1mm的线结,左例不予处理；对照组：暴露大鼠右侧坐骨神经,不进行处理,分层缝合切口,左侧不予处理。在手术后第3d,7d,10d,15d,20d,30d,40d时观察感觉异常和痛觉过敏阈值的变化,以触诱发痛阈值和热痛觉过敏闽值出现明显且稳定的下降为模型建立成功的标志。行为学观察结束后,冰上处死大鼠取L4-6DRG,Real-time PCR及Western blotting检测其CaV2.2转录水平及蛋白的表达变化。结果：CCI组较对照组手术后第3天即有MWT的下降(P<0.01),至术后第7天MWT及TWT达到高峰(P<0.01),之后在观察期内基本保持稳定。CCI组CaV2.2mRNA及蛋白水平较对照组表达明显增加(P<0.01)。结论：利用铬制肠线成功制备SD大鼠CCI模型,用于神经病理性疼痛的研究；CCI大鼠DRG中CaV2.2 mRNA及蛋白水平表达上调,提示CaV2.2与神经病理性疼痛的发生发展有关。第三部分鞘内注射LV-shCaV2.2e37a对CCI大鼠痛阈的影响及其对大鼠毒副作用的观察目的：研究鞘内注射LV-shCaV2.2e37a对CCI模型大鼠痛阈的影响,并观察其注射后的长期毒副作用。方法：健康雄性SD大鼠64只,随机分为5组,siRNA治疗组及阳性对照组14只,CCI组、阴性对照组及正常组各12只。siRNA治疗组于制作CCI模型第7天,大鼠出现明显的痛阈降低后,鞘内注射LV-shCaV2.2e 37a10μl(1×109TU／mL);阳性对照组于制作CCI模型后1鞘内置管,置管成功的大鼠于造模后第7天开始通过导管鞘内注射齐考诺肽(600ng／d)：阴性对照组于制作CCI模型后第7天鞘内注射空陵病毒颗粒10μl(1×109 TU／mL)；CCI组为CCI大鼠,不进行其它处理；正常组为正常大鼠。在鞘内注射后的3d,1w,2w,3w,4w,6w时分别观察大鼠的机械性缩足反射阈值(MWT)和CO2激光热缩足反射阈值(TWT)。于鞘内注射后1w,随机取2只siRNA治疗组大鼠处死取L4-6DRG,共聚焦显微镜观察DRG中GFP的表达,从而反映慢病毒载体在DRG组织细胞中的转染情况。大鼠于第6w行为学测定结束后处死,取心、肝、肺、肾、脊髓、脾,HE染色,常规病理观察LV-shCaV2.2e37a的毒副作用。结果：鞘内注射后3d,阳性对照组机械性缩足反射阈值(MWT)和CO2激光热缩足反射阈值(TWT)较同一时间点CCI组、siRNA治疗组及阴性对照组显著增高(P<0.01),较正常组无明显区别。鞘内注射后1w, siRNA治疗组大鼠MWT及TWT较同一时间点CCI组及阴性对照组明显提高(P<0.01),但仍低于阳性对照组及正常组(P<0.01)。鞘内注射后2w,siRNA治疗组大鼠MWT及TWT较同一时间点正常组及阳性对照组无显著差异(P>0.05),较CCI组及阴性对照组明显增高(P<0.01)。鞘内注射1w时病毒治疗大鼠DRG冰冻切片共聚焦显微镜检查显示GFP明显表达。鞘内注射LV-shCaV2.2e37a大鼠主要脏器在观察期内没有发现毒副作用。结论：鞘内注射LV-shCaV2.2e37a可显著提高CCI模型大鼠的机械性缩足反射阈值(MWT)和CO2激光热缩足反射阈值(TWT),同时观察期内其重要脏器未出现病理改变。更多还原

【Abstract】 N-type calcium channel is a pharmacological key target in treatment of chronic pain. N-type calcium channel inhibitors of common practice always cause side effects, such as the function of nonceciptice comductine pathway and centrol or auto neuro system. N-type calcium channels (CaV2.2) have two isoforms, CaV2.2e37a and CaV2.2e37b. CaV2.2e37a expressed particularly in DRG. Specific siRNA against CaV2.2e37a was searched and designed in our previous study, and the siRNA could specifically restrain the whole CaV2.2e37a gene expressing in 293FT cell. This project proposed to study on the analgesia effects of siRNA mediated by lentiviral vector in neuropathic pain model of CCI rats. Side effects were also observed. This study could provide new evidence to relieve neuropathic pain based on the target of CaV2.2e37a.PartⅠConstruction and identification of lentiviral vector of siRNA of CaV2.2e37a geneObjective:The lentiviral vector of RNA interference (RNAi) of CaV2.2e37a gene was constructed to study the neuropathic pain by restrainning the CaV2.2e37a gene expressing. Methods:The effective sequence of siRNA targeting CaV2.2e37a gene was confirmed in our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pLL3.7 vector by Hpa I and Xho I, which contained green fluorescent protein (GFP). The resulting vector containing CaV2.2e37a was confirmed by sequencing.293T cells were cotransfected with pLL3.7-CaV2.2e37a, pRsv-REV, pMDlg-pRRE and pMD2G. The titer of concentrated virus was tested by Real-time quantity PCR.Results:DNA sequence results showed that the RNAi vector of CaV2.2e37a (pLL3.7-CaV2.2e37a) was constructed successfully. The titer of concentrated virus was 1×109 Tu/ml. Conclusions:The lentivirus RNAi vector against CaV2.2 e37a was constructed successfully.PartⅡConstruction of CCI rats model and variation of CaV2.2 expression in CCI ratsObjective:Chronic constriction injury (CCI) rats pain model was constructed to investigate the variation of the expression of CaV2.2 in CCI rats" DRG. Methods:Twenty healthy male SD rats were randomly divided into two groups(n=10). In CCI group, rats were anesthetized with chloral hydrate (10%), the right sciatic nerve was exposed at mid-thigh level and four 4-0 chromic gut sutures were loosely ligated around the sciatic nerve approximately 1mm apart. The incision was closed with 4-0 silk suture. In control group, the right sciatic nerve was exposed as CCI group,then the incision was closed without any treatment. The variation of allodynia and thermal hyperalgesia were observed at stages of 3,7,10,15,20,30 and 40 days after ligation of the right sciatic nerve by Von-Frey filaments and CO2 laser. Successful rat model was set up when allodynia and thermal hyperalgesia was induced.40 days after the ligation of the sciatic nerve, rats were sacrificed by decapitation and the L4-6 dorsal root ganglias were dissected out on the ice. The variation of CaV2.2 mRNA and protein were tested by Real-time PCR and Western blotting. Results:The threshold of mechanical allodynia in right sides of rats in CCI group began to decrease 3 days after the ligation(P<0.01). Pain threshold came to peak 7 days after the ligation. The MWT and TWT of the CCI group rats were lower than the normal group rats(P<0.01). The mRNA level and protein level of CaV2.2 were significantly higher in CCI group than that of the control group (P<0.01). Conclusions:CCI pain model could be successfully constructed using chromic gut and it could be used for the research of neuropathic pain. Both of mRNA and protein expression level of CaV2.2 in CCI rats’DRG were increased, indicate that CaV2.2 is potentially involved in the neuropathic pain.Part III The observation of pain threshold alteration and side effects after intrathecal injection LV-shCaV2.2e37a in CCI ratsObjective:The effects and side effects of intrathecal injection of LV-shCaV2.2e37a were observed in CCI rats. Methods:Sixty-four healthy male rats were randomly divided into five groups, the siRNA therapy group and the active control group contained fourteen rats. Normal group, CCI group and the negative control group contained twelve seperately. In siRNA therapy group,CCI rats were intrathecally injected with 10μl LV-shCaV2.2e37a (1×109TU/mL) 7 days after the ligation of the sciatic nerve. In active control group, CCI rats were intrathecally injected with Ziconotide (600ng/d) 7 days after the ligation.In normal group was normal rats without any treatment. In CCI group were CCI rats without any other treatment. In negative control group,CCI rats were intrathecally injected with letiviral vector (1×109 TU/mL) without interfering sequence 7 days after the ligation. At various time points of 3d, 1w, 2w,3w,4w and 6w after intrathecal injection, pain threshold was tested. Two rats of the siRNA therapy group were sacrificed 1 week after intrathecal injection, L4-6 dorsal root ganglias were dissected which frozen sections observed by confocal laser scanning microscope. Rats were killed six weeks after injection. Heart, liver, lung, kidney, spine and spleen were taken out and HE staining slices of those organs were made to observe side effects of LV-shCaV2.2e37a intrathecal injection. Results:Pain threshold of rats in active control group was significantly higher than that of CCI group, siRNA therapy group and negative control group 3d after injection (p<0.01). After 1w injection, pain threshold of rats in siRNA therapy group was higher than that of CCI group and negative control group (P<0.01) while lower than that of normal group and active control group (P<0.01). Two weeks after injection, pain threshold of rats in siRNA therapy group was higher than that of CCI group and negative control group (P<0.01), had no significant difference with that of normal group and active control group (P>0.05). GFP was significantly expressed in DRG cells seven days after injection. No side effects of rats were observed after intrathecal injection of LV-shCaV2.2e37a. Conclusions:Pain of CCI rats could be alleviated for 6 weeks after intrathecal injection of LV-shCaV2.2e37a. Meanwhile, no side effects were observed throughout the whole process.更多还原