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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si NgR治疗组和生理盐水(NS)对照组各20只,HIBD组10只,si NgR治疗组脑室内注射si NgR及转染试剂10μl,NS对照组脑室内注射NS及转染试剂10μl,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,si NgR治疗组NgR c DNA条带不明显,NS对照组NgR c DNA条带清晰,两组管家基因GAPHD c DNA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;si NgR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si NgR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si NgR治疗组高于HIBD组和NS对照组(P<0.05),假手术组和si NgR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);si NgR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 investigate the effect of Nogo-66 receptor (NgR) -specific small interfering RNA (siRNA) on neural regeneration and function of neonatal rats after hypoxic-ischemic brain damage (HIBD). Methods Fifty neonatal HIBD models were established and randomly divided into three groups: si NgR treatment group and saline control group (n = 20), HIBD treatment group (n = 20), si NgR treatment group In the control group, intraperitoneal injection of NS and transfection reagent 10μl, HIBD group did not intraventricular injection; another 10 newborn rats were selected sham operation group, only the common carotid artery was separated, not ligated, without hypoxic-ischemic and ventricular Internal injection. The water maze test was used to analyze the escape time of rats. The expression of NgR and GAP - 43 protein in rat brain were analyzed by immunohistochemistry and image analysis. Results The results of RT-PCR gel electrophoresis showed that the NgRc DNA band of siNgR treatment group was not obvious, and the NgRc DNA band of NS control group was clear. The GAPHD c DNA bands of both groups were clear. Immunohistochemistry showed that more NgR immunoreactive product positive particles were seen in the NS control group; and the positive particles of the NgR immunoreactive product in the si NgR treatment group were significantly reduced. The number of positive cells of GAP-43 in sham operation group, HIBD group, NS control group and si NgR treatment group were (33.2 ± 1.3), (20.1 ± 1.2), (18.7 ± 1.4) and (28.1 ± 1.8) , Sham operation group and si NgR treatment group were higher than those in HIBD group and NS control group (P <0.05). There was no significant difference between sham operation group and si NgR treatment group (P> 0.05). The results of water maze test showed that the 3-day mean escape times of neonatal rats in HIBD group were (58.1 ± 10.3), (47.2 ± 10.1) and (42.5 ± 7.6) s respectively than those in sham operation group (34.2 ± 5.6 and 25.7 ± (37.5 ± 9.8), (29.1 ± 9.8) and (27.2 ± 9.3) s in the si NgR treatment group were significantly higher than those in the HIBD and NS control groups (P <0.05) (60.7 ± 5.2), (49.1 ± 9.9), (45.3 ± 9.3) s] were significantly shortened (P <0.05). Conclusion Chemosynthesis of specific siRNA can interfere with the expression of NgR in the brain tissue of neonatal rats, and to a certain extent, promote the regeneration of nerve and the recovery of neural function.