【作者】 李旭；

【导师】 黄宇光；

【作者基本信息】 北京协和医学院 ， 临床医学， 2009， 博士

【摘要】 目的：已有实验证明PSD-95与神经病理性疼痛密切相关,而RNA干扰(RNA interference, RNAi)技术是近年研究比较热门的治疗方向。本研究旨在利用RNAi技术沉默大鼠脊髓PSD-95基因,并观察此方法治疗神经病理性疼痛的效果。方法：将30只雄性SD大鼠随机分成6组(n=5),分别为空白对照,假手术组(3天和7天),以及CCI模型(3天、7天、14天)组,通过测量机械性痛阈和热痛阈来判断CCI模型是否成功。术后第3天、第7天、第14天分别处死大鼠,留取脊髓标本,进行real-time PCR分析PSD-95mRNA表达水平的变化。取不同重量大鼠建立鞘内置管给药模型,置管次日鞘内注射2%利多卡因,判断鞘内置管是否成功,并评价不同重量大鼠置管深度与效果的关系。选取合适重量大鼠,分组后(n=6)进行鞘内置管,鞘内给予PSD-95小干扰RNA(small interfering RNA, siRNA),最后一次给药后1天、3天、5天分别测量痛行为变化,并于1天和5天处死动物,留取脊髓标本,进行real-time PCR分析PSD-95siRNA表达的变化。选取合适重量大鼠,分组(n=5)后进行鞘内置管,置管后3-4天建立坐左侧骨神经CCI(chronic constriction injury,’慢性压迫性损伤)模型,并鞘内分别给予PSD-95siRNA、生理盐水、转染试剂(i-FectTM)、阴性对照siRNA,在给药后1天和5天分别测量痛阈,并处死动物,留取脊髓标本,进行real-time PCR分析PSD-95mRNA表达水平的变化。结果：大鼠CCI模型手术侧后足机械性痛阈和热痛敏阈值明显下降(P<0.05),但脊髓背角PSD-95mRNA表达无明显变化。正常大鼠中,鞘内注射PSD-95siRNA可降低大鼠脊髓背角PSD-95mRNA表达水平(P<0.05),但对其痛阈无显著影响。鞘内注射PSD-95siRNA可明显缓解CCI模型大鼠的神经病理性疼痛(P<0.05)。结论：CCI模型大鼠其脊髓背角PSD-95mRNA表达水平无显著改变,鞘内注射PSD-95siRNA可以明显缓解CCI模型大鼠的热痛觉过敏和机械性痛觉异常。更多还原

【Abstract】 Objective:It is taught that PSD-95is involved in the generation and development of chronic neuropathic pain. RNAi (RNA interference) technique and small interfering RNA (siRNA) now offer a powerful and highly specific tool for therapeutic gene silencing. The aim of this study is to investigate the efficiency of PSD-95gene silencing by siRNA, together with its therapeutic effect on chronic constriction injury model rats.Methods:Ninety-six male SD rats, weighting200-250g were randomly divided into6groups (n=5for each group):Naive, Sham(sham3d and sham7d), CCI(CCI3d, CCI7d and CCI14d). Mechanical allodynia and thermal hyperalgesia were measured to evaluate the success of the model. The rats were executed on3rdd、7thd、14thd after the surgery, and the level of PSD-95mRNA was compared among each group. Then the proper length of the catheter, rat body weight, and the agent dosage were preliminarily evaluated for intrathecal administration. PSD-95siRNA(2μg/d X4d), saline, i-FectTM or control siRNA was given intrathecally(i.t). Mechanical allodynia and thermal hyperalgesia were measured to evaluate the analgesic effect of PSD-95siRNA. On the1st day and5th day after the last administration, rats were sacrificed and lumbar spinal cords were sampled for further real-time PCR analysis.Results:Chronic constriction injury of rat sciatic nerve produced a clear-cut mechanical allodynia and thermal hyepralgesia in rats submitted to the surgery compared to the sham operated and naive ones. Intrathecal PSD-95siRNA (2μg/d X3d, i.t.) could significantly reduce the level of PSD-95mRNA in the spinal cord, and intrathecal PSD-95siRNA (2μg/dX4d, i.t.) could elevate the mechanical withdrawal threshold and paw withdrawal thermal latency of CCI rats without affecting the baseline antinociception.Conclusion:The PSD-95mRNA level exhibited no chage in the superficial dorsal horn after chronic constriction injury of rat sciatic nerve. In vivo delivery of PSD-95siRNA using i-FectTM could significantly silent the target gene, as relieved neuropathic pain in CCI model rats.更多还原