骨髓间充质干细胞移植在脑缺血损伤中的作用与生长相关蛋白-43表达的相关性

Marrow Stem Cells Transplantation and Its Correlation with Expression of Growth Associated Protein-43 in Rats with Cerebral Ischemia Injury

目的:探讨脑缺血/再灌注损伤后大鼠脑内介导骨髓间充质干细胞(MSCs)后神经组织修复和行为学变化的作用机制;观察生长相关蛋白-43(GAP-43)在神经元可塑性中的作用。方法:成年健康雄性Wistar大鼠118只分为假手术组42只,再灌注组42只及移植组30只,另4只大鼠为细胞的移植供体。将再灌注组及移植组成功制备为脑缺血再灌注损伤模型。移植组大鼠于造模3 d时颅内注射经培养纯化后的MSCs 10μl。造模后3、8、164、8 h及5、91、6 d时再灌注组及假手术组各取6只,移植组于8、164、8 h及5、91、6 d时取5只,观察凋亡细胞及GAP-43阳性细胞在皮质及海马区的表达;缺血半影区梗死灶的形成和移植细胞定向迁移的动态变化。结果:与再灌注组比较,移植组在移植后5 d梗死灶区MSCs及GAP-43阳性细胞数明显增加,9 d时达高峰,16 d时呈低水平表达;16 d时神经功能缺损程度评分明显降低(均P<0.05,0.01)。结论:MSCs脑内移植可介导缺血半影区梗死面积的修复;GAP-43表达抑制神经元凋亡,促进细胞的可塑性。

Objective： To explore the differentiation of the marrow stem cells（MSCs） in vitro,the mechanism of neural repair and behavioral change mediated by MSCs transplantation,and observe the role of growth associated protein-43 in neuronal plasticity following cerebral ischemia/reperfusion injury.Methods： 118 adult male Wister rats were divided randomly into sham-operation group（n=42）,cerebral ischemia/reperfusion group（n=42） and cell transplantation group（n=30）.According to the time points of reperfusion,each group was divided into 7 subgroups： 3 h,8 h,16 h,48 h and 5 d,9 d,16 d（n=6 each）.All ischemia duration was 1 h.MSCs transplantation was performed after the reperfusion at each time point（n=6）.Another 4 Wistar rats severed as donors of MSCs transplantation.Following reperfusion in each time point brain tissue was obtained.The MSCs were induced by basic fibroblast growth factor（bFGF） and epidermal growth factor（EGF） in vitro and identified as nerve-like stem cells,neuron-like cells,neurogliocyte-like cells by Nestin,5-bromodeoxyuridine（BrdU） and glial fibrillary acidic protein（GFAP） respectively.Neurological behavior detection and neurological scoring were performed respectively after MSCs transplantation.Modified suture method was used to establish the models of middle cerebral artery occlusion/reperfusion（MCAO/R）,and TUNEL method was applied to observe the distribution of apoptosis in cortex region and hippocampal region,and GAP-43 positive neurons were detected by immunohistochemistry in two regions.The formation of infarction in cerebral ischemic penumbra and the dynamic change of directed migration of transplanted cells were observed.Results： In the reperfusion 9 d group,the number of GAP-43 positive neurons reached the peak and was significantly increased as compared with sham operation group（P0.01）.In the reperfusion 16 d group,apoptosis rate reached the maximum（P0.01）.In the cell transplantation 5 d group,infarction area was significantly increased as compared with ischemia/reperfusion group（P0.01）.In the cell transplantation 9 d group,the number of transplanted neurons reached the peak（P0.01）.In the cell transplantation 16 d group,neural function score was obviously decreased（P0.01）.Conclusion： The repair of infarction area in cerebral ischemic penumbra is mediated by the transplantation of MSCs.The expression of GAP-43 inhibits apoptosis of neurons and promotes the plasticity of cerebral nerves.