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目的探讨突触后致密蛋白95(PSD-95)基因沉默对神经病理性疼痛模型大鼠脊髓Ca2+/钙调蛋白依赖的蛋白激酶Ⅱα(CaMKⅡα)表达及活性的影响。方法用化学合成针对大鼠PSD-95基因的siRNA转染神经母细胞瘤/大鼠神经胶质细胞瘤杂交瘤细胞(NG108-15细胞),并观察干扰效果。选取91只成年SD大鼠随机分为3组:Naive组、假手术组(sham)与坐骨神经慢性缩窄损伤(CCI)手术组。CCI组大鼠进一步被分为4个亚组,分别于CCI术后第5天开始鞘内注射生理盐水(Control组)、转染试剂(Vehicle组)、阴性对照(mmRNA组)和PSD-95基因特异siRNA(siRNA组)。连续给药3d,各组大鼠分别于鞘内给药后第1、3、7天评估机械缩足反射阈值(MWT)的改变,并留取腰4～6脊髓节段标本,以Western blotting方法观察脊髓背角PSD-95、CaMKⅡα蛋白表达水平及活性的变化。结果大鼠PSD-95基因特异的siRNA可以有效地沉默NG108-15细胞中PSD-95基因表达。与生理盐水治疗组相比,鞘内注射PSD-95特异siRNA 3d后,大鼠脊髓背角PSD-95蛋白水平明显降低(P<0.05),CCI大鼠神经病理性疼痛得到明显缓解(P<0.05),同时脊髓背角pThr286CaMKⅡα蛋白水平受到明显抑制(P<0.05),而总CaMKⅡα蛋白水平无明显变化(P>0.05)。结论 PSD-95基因特异的siRNA可被成功导入大鼠中枢神经系统,引起脊髓PSD-95基因沉默,在缓解病理性疼痛的同时可抑制神经损伤导致的脊髓CaMKⅡα磷酸化水平的增加,阻断中枢敏化相关的信号通路。
Objective To investigate the effect of silent PSD-95 gene silencing on Ca2 + / calmodulin-dependent protein kinase Ⅱ a (CaMKⅡα) expression and activity in neuropathic pain model rats. Methods The neuroblastoma / rat glioma cell hybridoma cells (NG108-15) were transfected with chemically synthesized siRNA targeting PSD-95 gene and the interference effect was observed. 91 adult SD rats were randomly divided into three groups: Naive group, sham group and CCI group. CCI rats were further divided into four subgroups: control group, Vehicle group, negative control group (mmRNA group) and PSD-95 Gene-specific siRNA (siRNA group). The rats in each group were sacrificed on days 1, 3 and 7 after intrathecal administration to assess the change of mechanical contractive threshold (MWT) Methods The changes of PSD-95 and CaMKⅡα protein expression in spinal dorsal horn were observed. Results Rat PSD-95 gene-specific siRNA effectively silence PSD-95 gene expression in NG108-15 cells. After intrathecal injection of PSD-95 specific siRNA for 3 days, PSD-95 protein level in spinal dorsal horn was significantly decreased (P <0.05), and neuropathic pain was significantly relieved in CCI rats (P <0.05 ), While pThr286CaMKⅡα protein level in spinal dorsal horn was significantly inhibited (P <0.05), while total CaMKⅡα protein level did not change significantly (P> 0.05). Conclusion PSD-95 gene-specific siRNA can be successfully introduced into the central nervous system of rat and cause PSD-95 gene silencing in the spinal cord. While relieving pathological pain, PSD-95 siRNA can inhibit the increase of phosphorylation of CaMKⅡα in spinal cord induced by nerve injury, Sensitization-related signaling pathways.