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目的探讨RNA干扰排斥性导向分子a(repulsive guidance molecule a,RGMa)对大鼠脑缺血再灌注损伤后大脑皮质血管再生的作用及其可能的机制。方法大鼠随机分为假手术组(S组)、脑缺血再灌注损伤组(I/R组)、脑缺血再灌注损伤+RGMa特异性RNA干扰重组腺病毒干预组(I/R+rAd-shRGMa组)以及脑缺血再灌注损伤+空载体重组腺病毒注射组(I/R+rAd-HK组)。大鼠在脑缺血前接受腺病毒注射,然后采用线栓法制备大鼠大脑中动脉缺血再灌注损伤模型。再灌注后2d免疫荧光双标法定位RGMa及其受体Neogenin在血管内皮细胞上的表达。腺病毒注射后7d,免疫组化标记CD31+的血管内皮细胞,计数大脑皮质缺血周围区微血管数;蛋白质印迹实验检测血管内皮生长因子a(VEGFa)蛋白表达水平;评估神经功能缺损。结果在缺血再灌注损伤后,RGMa及其受体Neogenin在CD31+细胞上均有表达。RNA干扰抑制RGMa表达后,微血管数量增多,VEGFa蛋白表达水平升高,神经功能缺损减轻。结论 RNA干扰RGMa表达可促进缺血再灌注损伤后缺血皮质周围区血管再生,该过程可能与脑组织VEGFa表达水平上调有关。
Objective To investigate the effect and possible mechanism of RNA interference targeting repulsive guidance molecule a (RGMa) on cerebral cortex angiogenesis after cerebral ischemia-reperfusion injury in rats. Methods Rats were randomly divided into sham-operation group (S group), cerebral ischemia-reperfusion injury group (I / R group), cerebral ischemia-reperfusion injury + RGMa specific RNA interference recombinant adenovirus intervention group rAd-shRGMa group) and cerebral ischemia-reperfusion injury + empty vector recombinant adenovirus injection group (I / R + rAd-HK group). Rats were injected with adenovirus before cerebral ischemia and then the middle cerebral artery occlusion (MCAO) model was established by thread occlusion. Two days after reperfusion, the expression of RGMa and its receptor Neogenin on vascular endothelial cells was detected by double immunofluorescence staining. Seven days after injection of adenovirus, endothelial cells labeled with CD31 + were immunohistochemically labeled, and the number of microvessels in the peri-ischemic area of the cerebral cortex was counted. The protein expression of VEGFa was detected by Western blotting, and the neurological deficits were evaluated. Results After ischemia-reperfusion injury, RGMa and its receptor Neogenin were expressed on CD31 + cells. RNA interference inhibited the expression of RGMa, the number of microvessels increased VEGFa protein expression increased neurological deficit reduced. Conclusion RNA interference of RGMa expression may promote angiogenesis in ischemic cortex surrounding ischemia / reperfusion injury, which may be related to the up-regulation of VEGFa expression in brain tissue.