水溶性脂聚体介导NR2B siRNA治疗神经病理性痛的实验研究

目的 探讨水溶性脂聚体(WSLP)介导含2B亚基的N-甲基-D-天冬氨酸受体小干扰RNA(NR2B siRNA)治疗大鼠神经病理性痛的可行性.方法 健康雄性SD大鼠100只,周龄6周,体重180～200 g,采用随机数字表法,将大鼠随机分为5组(n=20):对照组(C组)、假手术组(S组)、神经病理性痛组(NP组)、WSLP-NR2B siRNA组(W组)、WSLP-阴性对照NR2B siRNA(WN组).采用坐骨神经分支部分结扎法制备大鼠神经病理性痛模型.C组不予任何处理;S组仅暴露坐骨神经,不牵拉和损伤神经;NP组于制备模型后即刻鞘内注射生理盐水20μl;W组和WN组于制备模型后即刻分别鞘内注射相应的siRNA 20μl.于模型制备前1 d及制备后3、7、14和21 d时测定机械缩足反应阈值(MWT)及热缩足反应持续时间(TWD),模型制备后3 d,痛阈测定结束后,每组取10只大鼠,取L4-6节段背根神经节,测定NR2B mRNA及其蛋白的表达水平.结果 与S组比较,NP组、W组和WN组MWT降低,TWD延长,NR2B mRNA及其蛋白表达上调(P＜0.05或0.01),C组上述指标差异无统计学意义(P＞0.05);与NP组比较,W组MWT升高,TWD缩短,NR2B mRNA及其蛋白表达下调(P＜0.01),WN组上述指标差异无统计学意义(P＞0.05).结论 WSLP不仅成功介导NR2B siRNA,抑制NR2B的表达,还可减轻大鼠神经病理性痛.

Abstract：

Objective To investigate the feasibility of NR2B small interference RNA(NR2B siRNA)carried by water-soluble lipopolymer(WSLP)for treatment of neuropathic pain in rats.Methods One hundred healthy male SD rats weighing 180-200 g were randomly divided into 5 groups(n=20 each):normal control group (group C),sham operation group(group S),neuropathic pain group(group NP),group WSLP-NR2B siRNA (group W)and group WSLP-negative NR2B siRNA(group WN).Neuropathic pain was induced by partial ligation of sciatic nenre.WSLP-NR2B siRNA complex was formed by binding WSLP and NR2B siRNA.Normal saline.WSLP-NR2B siRNA complex and WSLP-negative NR2B siRNA 20μl were injected intrathecally after operation in NP,W and WN groups respectively.Mechanical withdrawal threshold(MWT)and thermal withdrawal duration (TWD)were measured before(baseline)and at 3,7,14 and 21 days after operation.Ten animals in each group were sacrificed on the 3rd day after operation and the lumbar segment(L4-6)of the dorsal root ganglia was removed for determination of the expression of NR2B mRNA and protein using RT-PCR and Western blot analysis.Results Sciatic nerve ligation significantly decreased MWT and prolonged TWD and increased NR2B mRNA and protein expression in group NP as compared with group C.WSLP-NR2B siRNA complex significantly reduced sciatic nerve ligation-induced hyperalgesia and decreased NR2B mRNA and protein expression in group W as compared with group NP.Conclusion WSLP not only mediates NR2B siRNA successfully and inhibits the expression of NR2B,but also reduces neuropathic pain in rats.     

鞘内注射水溶性脂聚体运载NR2B siRNA减轻坐骨神经缩窄性损伤大鼠神经病理性疼痛

脊髓背角N-甲基-D-天冬氨酸受体2B亚基（NR2B）的过度表达在神经病理性疼痛的产生和维持方面起关键作用,利用siRNA抑制NR2B的表达有望成为神经病理性疼痛甚至神经损伤治疗的新途径,然而,目前仍然缺乏NR2B siRNA安全且高效的载体。本实验探讨了低分子量聚乙烯亚胺和胆固醇组成的水溶性脂聚体（WSLP）运载NR2B siRNA治疗大鼠神经病理性疼痛的可行性,以寻找神经病理性疼痛基因治疗的安全且高效的载体。结果发现：WSLP与NR2B siRNA复合物（WSLP/siRNA）鞘内注射后3d,坐骨神经缩窄性损伤导致的神经病理性疼痛大鼠脊髓背角NR2B的基因表达显著下降,转录水平及蛋白水平的表达分别下降59%及54%（P < 0.01）,WSLP/乱序siRNA(scRNA) 及低分子量聚乙烯亚胺/siRNA鞘内注射后NR2B的基因表达无明显变化。WSLP/siRNA鞘内注射后3,7,14及21d神经病理性痛大鼠患足的疼痛程度明显减轻,而注射低分子量聚乙烯亚胺/ siRNA及WSLP/scRNA却无此作用。由此表明,WSLP可有效运载siRNA抑制神经病理痛大鼠NR2B基因的过度表达,从而对神经病理性疼痛大鼠具有治疗作用。