TERT 基因转染的 BMSCs 对血管性痴呆大鼠认知功能影响的分子机制研究

摘要

Objective To study the molecular mechanisms underlying the effects of bone marrow mesenchymal stem cells (BMSCs) transfected with telomerase reverse transcriptase (TERT) on the restoration of learning and memory ability and the impact on synaptic plasticity in the hippocampal CA 1 area of rats with vascular dementia (VD).Methods Rat BMSCs were isolated and identified , then transfected by pLXSN-TERT recombinant .Colony formation assay in soft agar was used to detect the tumorigenicity of TERT-BMSCs in vitro .Thirty-six male Wistar rats were randomly divided into the con-trol group, VD group, BMSCs group and TERT-BMSCs group.Two-vessel occlusion (2VO) was employed to make VD models .Morris water maze test was done for the detection of spatial learning and memory ability .Then the ultrastructures of synapses in the four groups were detected by transmission electron microscope .The expression of BDNF , NMDAR1 and SYN in hippocampal CA 1 region was determined by immunohistochemistry .Results Both Morris water maze test and transmission electron microscope showed that the behavior and morphology were improved in the BMSCs and TERT -BMSCs groups as compared with that of the VD group (all P<0.05), and the TERT-BMSCs group was better (all P<0.05). The average expression levels of BDNF , NMDAR1 and SYN protein in the BMSCs and TERT-BMSCs groups were higher than those in the VD group (all P<0.05), and TERT-BMSCs group had more significant effects than BMSCs group (all P<0.05).Conclusions TERT-BMSCs can more significantly improve the ability of spatial learning and memory in VD rats than regular BMSCs .This mechanism might be associated with enhancement of the expression of BDNF , NMDAR1 and SYN protein in hippocampal CA 1 region and affecting the synaptic plasticity .%目的：研究端粒酶逆转录酶（TERT）基因转染的骨髓基质干细胞（BMSCs）对血管性痴呆（VD）大鼠学习记忆功能的恢复及其对海马CA1区突触可塑性影响的分子机制。方法经大鼠股骨及胫骨分离并鉴定BMSCs，构建反转录病毒pLXSN-TERT重组子并转染BMSCs，软琼脂克隆形成实验检测体外培养的TERT-BMSCs的成瘤性。将36只雄性Wistar大鼠随机分为正常对照组、痴呆模型组、BMSCs组和TERT-BMSCs组。采用双侧颈总动脉永久性结扎方法制作VD大鼠模型，Morris水迷宫测试法检测各组学习记忆能力，透射电镜观察海马CA1区超微结构变化；免疫组织化学方法观察海马CA1区BDNF、NMDAR1、SYN蛋白表达。结果 Morris水迷宫测试及透射电镜观察均显示，BMSCs组、TERT-BMSCs组较痴呆模型组学习记忆能力、海马CA1区超微结构明显改善（P均＜0．05），且TERT-BMSCs组比BMSCs组改善更为明显（P均＜0．05）。 BMSCs组、TERT-BMSCs组BDNF、NMDAR1、SYN蛋白阳性细胞着色的平均灰度值比痴呆模型组增高（P均＜0．05），且TERT-BMSCs组比BMSCs组增高更为显著（P均＜0．05）。结论 TERT基因转染的BMSCs比普通BM-SCs对VD的康复治疗作用更为显著，其分子机制可能与增加大鼠海马CA1区BDNF、NMDAR1和SYN的表达，影响了突触可塑性有关。