GDNF基因修饰的神经干细胞抑制脑卒中后大鼠的Caspase-3表达

摘要

目的 研究胶质源性神经营养因子(glial cell line-derived neural factor,GDNF)基因修饰的神经干细胞(neural stem cells,NSCs)移植对脑卒中后大鼠缺血侧脑组织内半胱氨酰天冬氨酸特异性蛋白酶-3(cysteinyl aspartate specific proteinase-3,caspase-3)表达的影响,探讨GDNF基因修饰的神经干细胞(GDNF/NSCs)移植对大鼠脑卒中的神经保护作用机制.方法 取新生大鼠脑组织分离培养NSCs,收集第6代前的NSCs备用.用重组腺病毒GDNF转染神经干细胞,制备GDNF/NSCs.暂时性阻塞大鼠大脑中动脉制备脑卒中模型,3d后,用脑立体定位仪向卒中侧侧脑室分别给予NSCs、GDNF/NSCs和生理盐水.再灌注时间1周、2周、3周、5周、7周后处死大鼠(n=3).裂解卒中侧脑组织,离心后得到脑组织蛋白样品,通过蛋白免疫印迹( Western Blotting)检测Caspase-3表达.结果 GDNF/NSCs、NSCs、NS各组caspase-3的表达在1周、2周、3周、5周、7周各时间点均逐渐降低.NSCs组、GDNF/NSCs组显著低于NS组(P＜0.01；P＜0.001)；GDNF/NSCs组明显低于NSCs组(P＜0.01).结论 GDNF/NSCs移植治疗脑卒中的机制可能与抑制caspase-3表达有关.%Objective To study the effects of grafting neural stem cells( NSCs) modified by glial cell line-derived neural factor(GDNF) gene (GDNF/NSCs) on the expression of Caspase-3 in rats subjected to cerebral ischemia reperfusion. Methods NSCs were cultured from newborn rats and NSCs before 6 generation were used for grafting use. NSCs were infected by recombinant GDNF adenovirus to prepare NSCs - overexpressing GDNF( GDNF/NSCs). Rat stroke was performed by occluding the middle cerebral artery occlusion for 2 h and reperfusion. At 3 days after reperfusion, NSCs, GDNF/NSCs and saline was infused into ipsilateral ventricle respectively. According to different reperfusion time, each group was subdivide into 5 groups: 1,2,3,5,7 weeks ( n = 3 ). At each time point, rats were sacrificed and brains were removed. Detect the expression of caspase-3 by Western Blotting. Results The expressions of caspase-3 in all the three groups gradually were decreased at given time point. NSCs group and GDNF/NSCs group significantly decreased caspase-3 compared with ischemic group(P ＜0. 01 ;P ＜0. 001). Besides, The GDNF/NSCs group showed more efficient than NSCs group(P＜0. 01). Conclusion Neuroprotection of the grafting GDNF/NSCs may be realted to inhibit the expression of caspase-3.