脑脉通联合骨髓间充质干细胞移植对脑缺血大鼠神经元突触重建的影响

Influence of Naomaitong on neuron synapse reconstruction of BMSCs transplanting in rats with cerebral ischemia

目的观察BMSCs移植后脑缺血再灌注大鼠神经元结构变化及突触重建标志性蛋白的变化特点及脑脉通对其的影响,探讨脑脉通联合BMSCs移植促进脑缺血神经功能修复的作用途径。方法体外全骨髓贴壁筛选培养法扩增BM-SCs;线栓法制备脑缺血模型;大鼠脑缺血再灌注后24h颈内动脉移植BMSCs;脑脉通灌胃给药;BMSCs移植后7d、14d、28d综合测评神经功能,透射电镜观察神经元突触结构,免疫组织化学法测定神经丝蛋白(NF-200)和生长相关蛋白(GAP-43)及突触素蛋白(Synaptophysin,Syn)表达变化。结果模型各组神经功能评分均较假手术组降低;脑脉通和联合组各时间点、移植28d组神经评分较模型组增高;联合7d和28d组评分较移植组增高。模型各组NF-200和GAP-43及Syn表达均较假手术组减弱;与模型组比较,脑脉通和移植28d组、联合14d和28d组大鼠NF-200、GAP-43、Syn表达均明显增强;与移植组比较,联合14d和28d组的NF-200和GAP-43表达均增强,28d组Syn表达增强显著。结论脑缺血引起的神经元结构损伤随再灌注时间延长呈加重趋势,并使突触重塑功能减弱;BMSCs移植脑早期对神经功能修复作用不明显,脑脉通可使BMSCs移植后修复神经功能的作用提前并增强,其作用途径可能与增强突触重建相关生长蛋白GAP-43水平,进而促进神经元突触重建相关。

Objective To observe the changes of neuron structure and the identification marker proteins of synapse reconstruction,as well as the influence of Naomaitong(NMT)on the changes,then to explore the way of combination of NMT and BMSCs transplantation in promoting the restoration of nervous function. Methods Rats whole bone marrow were cultivated and BMSCs were purified and increased by methods of adherence and selection in vitro.Middle cerebral artery occlusion(MCAO) model was duplicated with nylon thread.Rats of transplantation and combination groups were transplanted with BMSCs via carotid artery at 24 h after cerebral ischemia reperFusion.NMT was used by intragastric administration.Rats general neural function(GNF) were measured and neurons synapse structure was observed by using transmission electron microscopy,then the expression of NF-200,GAP-43 and Syn were determined by using immunohistochemical method at 7d(earlier period),14d(metaphase)and 28d(anaphase)after BMSCs transplantation as the specific identification marker of neuron structure and synapse reconstruction. Results Rats GNF in each model group was lower than that in sham operation group.Rats GNF in each NMT and combination group,28d transplantation group all increased in comparison with that of model groups.Rats GNF in 7d and 28d combination groups was higher than that in transplantation groups.Rats NF-200,GAP-43 and Syn in each model group was lower than that in sham operation group.Comparing with model groups,the expression of NF-200,GAP-43 and Syn in NMT and transplantation 28d,14d and 28d combination groups all increased obviously.Rats' expression of NF-200 and GAP-43 in 14d and 28d,and Syn in 28d combination groups enhanced obviously than that in transplantation groups. Conclusion Rats neuron structure was damaged obviously being caused by cerebral ischemic injury,and appeared the progressively enhanced tendency following the prolongation of reperfusion,while the synapse reconstruction attenuated.BMSCs transplantation showed no significant effect on nervous function restoration in earlier period.NMT could make the effect of BMSCs transplantation on GNF recovery ahead of schedule and enhance the effects of BMSCs transplantation.The way of combination administration might be related to up-regulation of GAP-43 and then to improve the neural synapse reconstruction.