RNA干扰抑制NgR表达对脑梗死大鼠皮质脊髓束可塑性的影响

RNA interference mediated down-regulation the expression of NgR promotes functional recovery and corticospinal tract plasticity in rats after cerebral infarction

目的研究应用RNA干扰技术下调Nogo受体(NgR)基因的表达对脑梗死大鼠神经功能恢复及皮质脊髓束可塑性的影响。方法线栓法制作左侧大脑中动脉闭塞脑梗死大鼠模型,用携带U6启动子和NgR特异短发夹RNA(shRNA)编码序列的质粒pNgR-shRNA1、pNgR-shRNA2行缺血侧侧脑室注射给药,以含非特异性shRNA编码序列的无关质粒pGenesil-Con(pCon)注射组及模型组作为对照,应用改良的神经功能缺损评分(mNSS)进行神经功能评定,Western blot检测梗死灶周围脑组织NgR的表达,免疫组织化学技术检测梗死灶周围脑组织生长相关蛋白43(GAP-43)的表达,生物素化葡聚糖胺(BDA)顺行示踪法观察皮质脊髓束可塑性变化。结果 pNgR-shRNA1组、pNgR-shRNA2组mNSS评分明显低于pCon组及模型组(P<0.05),pCon组及模型组mNSS评分差异不明显(P>0.05)。与pCon组及模型组相比,pNgR-shRNA1组、pNgR-shRNA2组梗死灶周围脑组织NgR的表达显著降低(P<0.05),GAP-43的表达明显增强(P<0.05),BDA顺行示踪见病变侧大脑脚水平及病变对侧C1-3脊髓水平BDA阳性纤维数量明显增多(P<0.05)。结论 RNA干扰抑制NgR表达能促进脑梗死大鼠皮质脊髓束可塑性改变及神经功能恢复。

Objective To explore the effects of down-regulation of Nogo receptor by RNA interference on functional recovery and corticospinal tract plasticity in rats after cerebral infarction. Methods The cerebral infarction model of left middle cerebral artery occlusion （MCAO） was established with left middle cerebral ar- tery suture occlusion method. The plasmid pNgR-shRNA1 or pNgR-shRNA2 carrying U6 promoter and NgR specific short hairpin RNA （shRNA） coding sequence was injected into rats＂ lateral ventricle which is ipsilateral to cerebral infarction. As control, the rats in other two groups were intraeerebroventricular injected with unre- lated plasmid pGenesil-Con （pCon） containing non-specific shRNA （pCon group） or had no intervention （mod- el group）. Modified neurological severity score （mNSS） was adopted to evaluate neural function of rats on the first day and 14th day after infarction. The NgR protein level in brain around cerebral infarction focal was de- tected by western blot. Immunohistochemistry technique was used to detect the expression of growth-associat- ed protein 43 （GAP-43）. Biotinylated dextran amine （BDA） anterograde tracer method was used to observe the plasticity changes of corticospinal tract. Results The mNSS score in pNgR-shRNAa and pNgR-shRNA2 group were significantly lower than pCon group and model group on 7 d, 14 d after infarction （P^0. 05）. The expression of NgR in cortex around infarction area in pNgR-shRNA1 and pNgR-shRNA2 group were much lower than pCon group and model group （P〈0. 05）. The number of GAP-43 positive cells in pNgR-shRNA1 and pNgR-shRNA2 group were more than pCon group and model group （P〈0. 05）. The number of BDA-pos- itive fibers in level of cerebral peduncle ipsilateral to the lesion and in level of C1-3 spinal cord contralateral to the lesion significantly increased in pNgR-shRNA1 and pNgR-shRNA2 group （P%0. 05）. Conclusions RNA interference mediated down-regulation the expression of NgR may promote functional recovery and enhances corticospinal tract plasticity in rats after cerebral infarction