协和医学杂志

2011, v.2(04) 343-349

[打印本页] [关闭]
本期目录(Current Issue) | 过刊浏览(Past Issue) | 高级检索(Advanced Search)

突触后致密物蛋白质95基因沉默及对神经病理性疼痛大鼠疼痛行为的干预
Small Interfering RNA-mediated Selective Knockdown of Postsynaptic Density Protein 95 Reverses Mechanical Allodynia in Neuropathic Pain Rats

李旭;申乐;许力;刘薇;虞雪融;黄宇光;
LI Xu,SHEN Le,XU Li,LIU Wei,YU Xue-rong,HUANG Yu-guang Department of Anesthesiology,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100730,China

摘要(Abstract):

目的评估小干扰RNA(small interfering RNA,siRNA)对突触后致密物蛋白质95(postsynaptic density protein95,PSD-95)基因的沉默效率及PSD-95基因沉默对谷氨酸诱导的神经细胞毒性与信号转导的影响,观察用RNA干扰(RNA interference,RNAi)技术沉默大鼠脊髓PSD-95基因治疗神经病理性疼痛的效果。方法用神经母细胞瘤/大鼠神经胶质细胞瘤杂交瘤细胞(NG108-15细胞)筛选大鼠PSD-95基因特异的siRNA,并用谷氨酸刺激PSD-95基因沉默的NG108-15细胞,检测细胞生长活力与信号通路蛋白质表达与磷酸化的改变。建立大鼠神经病理性疼痛的坐骨神经慢性压迫(chronic constrictioni njury,CCI)模型,预防性及治疗性鞘内给予PSD-95siRNA,留取脊髓标本,实时定量PCR(real-time PCR,RT-PCR)分析PSD-95 mRNA表达水平,并测定大鼠后足机械痛阈及热痛阈的变化。结果序列特异性最高的siRNA在最佳转染条件下使PSD-95基因表达水平下降91.5%;PSD-95基因沉默既可增强神经细胞对谷氨酸毒性的耐受能力,又可抑制Ca2+/钙调蛋白依赖的蛋白质激酶IIα(CaMKIIα)异构型磷酸化。正常大鼠鞘内注射PSD-95siRNA可降低大鼠脊髓背角PSD-95mRNA的表达水平38%(P<0.05),但对痛阈无显著影响;CCI模型大鼠鞘内注射PSD-95 siRNA可明显缓解神经病理性疼痛。结论 PSD-95基因在神经病理性疼痛的发生中起重要作用。鞘内注射PSD-95siRNA可以显著缓解CCI模型大鼠机械性和热痛觉过敏,PSD-95 siRNA可能成为有效控制神经病理性疼痛的基因治疗方法。
Objective Postsynaptic density protein 95(PSD-95) gene specific siRNAs were introduced in vitro or in vivo to determine its succedent influence on neuropathic pain relief,neuron viability and postsynaptic calcium/calmodulin-dependent protein kinase II(CaMKII) phosphorylation.Methods siRNAs of rat PSD-95 gene were synthesized for in vitro transfection into NG108-15 neuronal cells.The siRNA to PSD-95,or a mismatch RNA,was mixed with the transfection reagent,i-FectTM(vehicle),and delivered as repeated daily bolus doses(2 μg) via implanted intrathecal catheter to the lumbar spinal cord of rats which received chronic constriction injury(CCI)of sciatic nerve.Both mechanical allodynia and thermal hyperalgesia were measured on the post-operative day 3 and 7.PSD95 gene silenced NG108-15 cells were further stimulated by glutamate,with either the cell viability or the expression and phosphorylation of CaMKIIα to be assayed.Results The siRNAs suppressed PSD-95 gene expression level by 91.5% under the appropriate condition in vitro.High level glutamate induced neurotoxicity,which was partially eliminated by PSD-95 gene silencing.Glutamate also induced the postsynaptic expression and phosphorylation of CaMKIIα.PSD-95 siRNA injected intrathecally in normal rats lowered the expression level of PSD-95 mRNA at cornu dorsale medullae spinalis by 38%(P<0.05) but had no remarkable effect on the pain threshold.Intrathecal injection of PSD-95 siRNA remarkably alleviated neuropathic pain.Conclusions PSD-95 gene plays an important role in the development of neuropathic pain.Intrathecal injection of PSD-95 siRNA can remarkably alleviate mechanical and thermal hyperalgesia.PSD-95 siRNA may be a promising gene therapy for neuropathic pain.

关键词(KeyWords): 神经病理性疼痛;突触后致密物蛋白质95;谷氨酸兴奋性中毒;Ca2+/钙调蛋白依赖的蛋白质激酶IIα;慢性压迫损伤;小干扰RNA
neuropathic pain;postsynaptic density protein 95;glutamate excitotoxicity;calcium/calmodulin-dependent protein kinase II;chronic constriction injury;small interfering RNA

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(30672029);; 中央保健专项基金(B2009B076)

作者(Author): 李旭;申乐;许力;刘薇;虞雪融;黄宇光;
LI Xu,SHEN Le,XU Li,LIU Wei,YU Xue-rong,HUANG Yu-guang Department of Anesthesiology,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100730,China

Email:

DOI:

参考文献(References):

扩展功能
本文信息
服务与反馈
本文关键词相关文章
本文作者相关文章
中国知网
分享