解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (2 ): 170-175.doi: 10.3969/j.issn.0529-1356.2013.02.005

• 神经生物学 • 上一篇    下一篇

鞘内注射PSD-95 siRNA对神经病理性疼痛大鼠脊髓CaMKⅡα活性的影响

许力1 喻洁2 申乐1 李旭1 黄宇光1*   

  1. 1. 中国医学科学院北京协和医学院北京协和医院麻醉科，北京 100730; 2. 四川大学华西临床医学院麻醉科，成都 610041
  • 收稿日期:2012-12-07 修回日期:2012-12-27 出版日期:2013-04-06 发布日期:2013-04-06
  • 通讯作者: 黄宇光 E-mail:pumchhyg@yahoo.com.cn
  • 基金资助:

    中央保健专项基金（B2009B076）;国家自然科学基金资助项目

Influence of intrathecal siRNA of postsynaptic density protein 95 on CaMKⅡα activation in spinal cord of rats with neuropathic pain

XU Li1  YU Jie2  SHEN Le1  LI Xu1  HUANG Yu-guang 1*   

  1. 1. Department of Anesthesiology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730，China; 2. Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu 610041，China
  • Received:2012-12-07 Revised:2012-12-27 Online:2013-04-06 Published:2013-04-06

摘要：

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pThr286CaMKⅡα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。

关键词: 神经病理性疼痛 , 突触后致密蛋白95 , Ca2+ /钙调蛋白依赖的蛋白激酶Ⅱ&alpha, 慢性压迫损伤 , 小干扰RNA , RNA干扰 , 免疫印迹法 , 大鼠

Abstract:

Objective To explore the influence of intrathecal postsynaptic density protein 95(PSD-95) gene specific siRNAs on CaMKⅡα phosphorylation in spinal cord of rats with neropathic pain. Methods The NG108-15 neuroal cells were transfected with chemically synthesized siRNA targeting PSD-95 for comprehensive evaluation of its silence efficacy. Ninety-one adult male Sprague-Dawley rats were randomly divided into three groups: naive, sham and sciatic nerve chronic constriction injury (CCI) group. All the rats in CCI group were subdivided into four groups, which were treated with normal saline（control group）, transfection vehicle（vehicle group）, mismatch siRNAs（mmRNA group）and PSD-95 gene specific siRNAs（siRNA group）respectively. All the subgroup received corresponding agents intrathecally for 3 days, as was started 5 days after the chronic constriction injury of sciatic nerve. Mechanical withdrawal threshold (MWT) was measured on post intrathecal infusion day 1, 3 and 7 by a series of von Frey hairs. The rats were sacrificed at different times after intrathecal infusion to examine PSD-95 and phosphorylation of CaMKⅡα in the lumber 4-6 spinal cord by Western blotting. Results The siRNAs, which was exclusively identical to PSD-95, decreased PSD-95 level significantly in NG108-15 cells. The MWT of neuropathic pain rats increased significantly after intrethecal administration of PSD-95 specific siRNA ( P <0.05), compared with intrethecal injection of saline. Total CaMKⅡα level did not change after PSD-95 siRNA infusion（ P >0.05）, however, the pThr286 CaMKⅡα level on the spinal cord of the CCI rats decreased significantly ( P <0.05). Conclusion Administration of PSD-95 gene specific siRNAs attenuates the phosphorylation of CaMKⅡα Thr286 and central sensitization signaling cascades and results in the relief of neuropathic pain.

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