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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

李旭 申乐 许力 刘薇 虞雪融 黄宇光

李旭, 申乐, 许力, 刘薇, 虞雪融, 黄宇光. 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[J]. 协和医学杂志, 2011, 2(4): 343-349. doi: 10.3969/j.issn.1674-9081.2011.04.012
引用本文: 李旭, 申乐, 许力, 刘薇, 虞雪融, 黄宇光. 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[J]. 协和医学杂志, 2011, 2(4): 343-349. doi: 10.3969/j.issn.1674-9081.2011.04.012
Xu LI, Le SHEN, Li XU, Wei LIU, Xue-rong YU, Yu-guan HUANG. Small Interfering RNA-mediated Selective Knockdown of Postsynaptic Density Protein 95 Reverses Mechanical Allodynia in Neuropathic Pain Rats[J]. Medical Journal of Peking Union Medical College Hospital, 2011, 2(4): 343-349. doi: 10.3969/j.issn.1674-9081.2011.04.012
Citation: Xu LI, Le SHEN, Li XU, Wei LIU, Xue-rong YU, Yu-guan HUANG. Small Interfering RNA-mediated Selective Knockdown of Postsynaptic Density Protein 95 Reverses Mechanical Allodynia in Neuropathic Pain Rats[J]. Medical Journal of Peking Union Medical College Hospital, 2011, 2(4): 343-349. doi: 10.3969/j.issn.1674-9081.2011.04.012

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

doi: 10.3969/j.issn.1674-9081.2011.04.012
基金项目: 

国家自然科学基金 30672029

中央保健专项基金 B2009B076

详细信息
    通讯作者:

    黄宇光 电话:010-65295580, E-mail:pumchhyg@yahoo.com.cn

  • 中图分类号: R34;R363.1

Small Interfering RNA-mediated Selective Knockdown of Postsynaptic Density Protein 95 Reverses Mechanical Allodynia in Neuropathic Pain Rats

More Information
  • 摘要:   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 protein 95, 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 constriction injury, 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-95 siRNA可降低大鼠脊髓背角PSD-95 mRNA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-95 siRNA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。
  • 图  1  谷氨酸诱发的神经兴奋性毒性

    siRNA:小干扰RNA; APV: D (-)-2-Amino-5-phosphonopentanoic acid; siR-N:非沉默siRNA; siR-P: PSD-95基因特异性siRNA
    与Naïve比较,†P<0. 05;与siR-N比较,‡P<0. 05;与APV比较,* P<0. 05

    图  2  总CaMKIIα及磷酸化CaMKIIα Western blot分析

    N:阴性对照; 与阴性对照比较,†P<0. 05;与siR-N比较,‡P<0. 05;与APV比较,* P<0. 05
    siR-N:同图 1

    图  3  PSD-95基因沉默对CCI大鼠机械和热痛觉过敏的影响

    siR-N、siR-P:同图 1; 与非siR-P组比较,* P<0. 05

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出版历程
  • 收稿日期:  2011-08-07
  • 刊出日期:  2011-10-30

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