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目的:探讨大鼠脑缺血/再灌注损伤后骨髓间基质干细胞颅内移植及生长相关蛋白-43(GAP-43)表达在中枢神经再生中的作用机制。方法:成年健康雄性Wistar大鼠260只,随机分为对照组、假手术组各6只,再灌注组48只。再灌注组采用线栓法制备大鼠脑缺血/再灌注模型,根据缺血/再灌注时间细分为再灌注3h、8h、16h、24h、3d、7d、19d和21d8个时间点各6只,缺血时间均为1h;细胞移植随机分为对照组和移植组各36只,移植时间细分为16h、24h、3d、7d、19d和21d6个时间点各6只;跑台运动训练随机分为模型组和运动组各24只,训练时间细分为3d、7d、19d和21d4个时间点各6只;水迷宫试验随机分为对照组、再灌注组和康复组各24只,训练时间细分为3d、7d、19d和21d4个时间点各6只。另4只大鼠作为细胞移植的供体。采用免疫组织化学方法检测TUNEL和GAP-43在皮层区、海马齿状回阳性细胞中的表达,用TTC法观察皮层及海马梗死灶的改变。按照Bederson评分标准进行神经行为检测及功能评分。结果:缺血/再灌注3h点TUNEL阳性细胞增加,24h点达高峰,21d时仍维持较高水平表达;GAP-43阳性细胞缺血/再灌注3h点在两区表达增加,19d点达高峰;21d时降至最低。梗死灶于再灌注8～16h点开始逐渐形成,3d点梗死面体积最大,随后梗死面积逐渐缩小,21d时恢复趋于正常。再灌注组缺血8h～16h点行为症状加重,24h～19d点症状逐渐改善,21d时症状恢复到正常水平。结论:脑缺血后骨髓间充质干细胞移植激发GAP-43特异性表达有效地促进了中枢神经再生,并介导了神经功能的恢复。
Objective: To explore the mechanism of intracranial transplantation of bone marrow stromal stem cells and the expression of growth-associated protein-43 (GAP-43) in central nervous system regeneration after focal cerebral ischemia / reperfusion injury in rats. Methods: Two hundred and sixty healthy male Wistar rats were randomly divided into control group, sham-operated group, and reperfusion group. The model of cerebral ischemia / reperfusion was established by thread occlusion in reperfusion group. According to the ischemia / reperfusion time, the rats were subdivided into 6, 8, 16, 24, 3d, 7d, 19d and 21d Only, ischemic time were 1h; cell transplantation were randomly divided into control group and transplantation group of 36, the transplantation time subdivided into 16h, 24h, 3d, 7d, 19d and 21d 6 time points 6 each; treadmill training The rats were randomly divided into model group and exercise group with 24 rats each. The training time was divided into 6 groups at 3d, 7d, 19d and 21d respectively. The water maze test was randomly divided into control group, 24 reperfusion group and rehabilitation group, The training time was subdivided into 6 time points of 3d, 7d, 19d and 21d respectively. Another 4 rats served as donor of cell transplantation. Immunohistochemistry was used to detect the expression of TUNEL and GAP-43 in cortical and hippocampal gyrus-positive cells, and the changes of cortical and hippocampal infarction were observed by TTC method. Bederson score according to the standard of neurobehavioral testing and functional score. Results: The number of TUNEL positive cells increased at 3 h after ischemia / reperfusion, reached the peak at 24 h and remained high at 21 d. The expression of GAP-43 positive cells increased in both regions at 3 h after ischemia / reperfusion and peaked at 19 d ; 21d to a minimum. The infarct size gradually formed at 8-16h after reperfusion, and the infarct surface area at 3d was the largest. The area of infarction gradually decreased afterwards and recovered to normal at 21d. After reperfusion, the behavioral symptoms were aggravated at 8h to 16h after ischemia. Symptoms gradually improved at 24h to 19d and recovered to normal levels on day 21. CONCLUSION: Specific expression of GAP-43 stimulated by bone marrow mesenchymal stem cell transplantation after cerebral ischemia effectively promotes CNS regeneration and mediates the recovery of neurological function.