Nogo-66受体特异性小干扰RNA对缺氧缺血性脑损伤新生大鼠神经再生功能的影响

摘要

目的 探讨化学合成Nogo-66受体(NgR)特异性小干扰RNA(siRNA)对新生大鼠缺氧缺血性脑损伤(HIBD)后神经再生和功能的影响.方法 建立新生大鼠HIBD模型50只,随机分为siNgR治疗组和生理盐水(NS)对照组各20只,HIBD组10只,siNgR治疗组脑室内注射siNgR及转染试剂10 μl,NS对照组脑室内注射NS及转染试剂10 μl,HIBD组不做脑室内注射;另外选择10只新生大鼠为假手术组,只分离颈总动脉,不结扎,不做缺氧缺血处理和脑室内注射.应用水迷宫实验分析大鼠逃生时间,并用免疫组化法结合图像分析大鼠脑组织 NgR 及生长相关蛋白(GAP)-43蛋白表达水平.结果 RT-PCR凝胶电泳结果显示,siNgR治疗组NgR cDNA条带不明显,NS对照组NgR cDNA条带清晰,两组管家基因GAPHD cDNA条带均清晰.免疫组化显示NS对照组可见较多NgR免疫反应产物阳性颗粒;siNgR治疗组NgR免疫反应产物阳性颗粒明显减少.假手术组、HIBD组、NS对照组和siNgR治疗组GAP-43 免疫组化阳性细胞数分别为(33.2 ± 1.3)、(20.1±1.2)、(18.7±1.4)和(28.1±1.8)个,假手术组和siNgR治疗组高于HIBD组和NS对照组(P＜0.05) ,假手术组和siNgR治疗组差异无统计学意义(P＞0.05).水迷宫实验结果显示, HIBD组新生大鼠3 天平均逃生时间[ (58.1 ± 10.3)、(47.2 ± 10.1)、(42.5 ± 7.6) s ]较假手术组[(34.2±5.6)、(25.7±6.2)、(21.2±8.1)s]明显延长(P＜0.05);siNgR治疗组[(37.5±9.8)、(29.1 ±9.8)、(27.2±9.3)s]较HIBD组和NS对照组[(60.7±5.2)、(49.1±9.9)、(45.3±9.3)s]明显缩短(P＜0.05).结论 化学合成特异性siRNA能够干扰新生大鼠脑组织NgR表达,并在一定程度上促进大鼠神经再生和神经功能恢复.%Objective To study the effect of chemically synthesized Nogo-66 receptor ( NgR ) specific small interfering RNA ( siRNA) on nerve regeneration and function of newborn rats after hypoxic-ischemic brain damage ( HIBD) . Methods A total of 50 HIBD newborn rats were set up. They were randomly assigned OR allocated into siNgR group ( n=20 ) , normal saline ( NS ) group ( n=20 ) and HIBD group ( n=10 ) . The rats of siNgR group were given intraventricular injection of siNgR and transfection reagents ( 10μl ); the rats of NS group were given intraventricular injection of NS and transfection reagents (10μl);and the rats of HIBD group had no intervention. In addition to these three groups, there is another group, sham-operated group ( n=10 ) . The rats of sham-operated group were sham-operated ( common carotid artery was isolated but not ligated) and did not receive hypoxia-ischemia processing and intraventricular injection. Utilize water maze experiment to analyze the rats' escape time. The levels of NgR and GAP-43 protein in rats' brains were measured by immunohistochemistry and image analysis. Results RT-PCR gel electrophoresis results showed that the NgR cDNA stripe of siNgR group was not obvious, but the stripe of NS group was clear. At the same time, the GAPHD cDNA bands of the above two groups were both clear. There were more NgR positive immune reaction products ( brown particles) in NS group than in siNgR group. The number of GAP-43 positive cells by immunohistochemistry in sham-operated group, HIBD group, NS group and siNgR group was (33. 24±1. 32), (20. 14±1. 24), (18. 73±1. 41) and (28. 06±1. 78), respectively. The number of sham-operated group and siNgR group was greater than HIBD group and NS group ( P＜0. 05 ) . There was no statistical significant difference for the number of GAP-43 positive cells between sham-operated group and siNgR group ( P＞0. 05 ) . Water maze experiment results showed that the newborn rats ' average escape time ( s ) of HIBD group ( 58. 1 ± 10. 3,47. 2±10. 1, 42. 5±7. 6) was obviously longer than sham-operated group (34. 2±5. 6, 25. 7±6. 2, 21. 2±8. 1), and the difference was statistically significant (P＜0. 05). However, the average escape time of siNgR group (37. 5±9. 8, 29. 1±9. 8, 27. 2±9. 3) was obviously shorter than HIBD group and NS group (60. 7±5. 2, 49. 1±9. 9, 45. 3±9. 3), (P＜0. 05). Conclusions Chemically synthesized specific siRNA had the potential to interference the expression of NgR in the brain of newborn rats, and to a certain extent, could promote the nerve regeneration and neural functional recovery of rats.