黄芪甲苷配伍三七总皂苷对OGD/R大鼠骨髓间充质干细胞增殖、凋亡、迁移及神经分化的影响

Effects of astragaloside Ⅳ combined with Panax notoginseng saponins on proliferation, apoptosis, migration and neuronal differentiation of bone marrow mesenchymal stem cells in OGD/R model rats

目的观察黄芪甲苷(ASTIV)配伍三七总皂苷(PNS)对氧糖剥夺后再复糖复氧(OGD/R)大鼠骨髓间充质干细胞(BMSCs)增殖、凋亡、迁移及神经分化的影响。方法全骨髓贴壁法分离、培养、扩增、纯化BMSCs,流式细胞仪检测BMSCs表面标志物CD29、CD90、CD34、CD45阳性表达率。取第3代BMSCs,采用AST IV与PNS高(100μmol/L+60μmol/L)、中(50μmol/L+30μmol/L)、低(25μmol/L+15μmol/L)剂量配伍预处理24 h,以OGD/R建立缺血再灌注损伤模型,同时设立对照组和模型组。CCK-8法测定细胞增殖情况,流式细胞术检测细胞凋亡,Transwell实验检测细胞迁移,巢蛋白(Nestin)/神经元特异性烯醇化酶(NSE)、Nestin/胶质纤维酸性蛋白(GFAP)免疫荧光双标观察BMSCs向神经元及星形胶质细胞分化情况。结果成功培养分离BMSCs,流式细胞仪检测CD29、CD90阳性率分别为94.23%、94.69%;而CD34、CD45阳性表达率分别为5.76%、5.31%;与对照组比较,模型组细胞存活数量明显减少(P<0.05)、细胞凋亡率显著增加(P<0.05),与模型组比较,AST IV与PNS不同剂量配伍均能促进BMSCs增殖(P<0.05、0.01),并抑制细胞凋亡(P<0.05、0.01);与对照组比较,模型组、AST IV与PNS不同剂量配伍均能促进BMSCs迁移(P<0.05),与模型组比较,AST IV与PNS不同剂量配伍组的迁移细胞数量明显增加(P<0.05);与对照组比较,模型组与AST IV与PNS不同剂量配伍组均能促进BMSCs向神经元及星形胶质细胞分化(P<0.01),与模型组比较,ASTIV与PNS不同剂量配伍组的Nestin/NSE、Nestin/GFAP阳性表达率明显增高(P<0.01)。结论ASTIV配伍PNS在体外能促进缺血再灌注模型BMSCs增殖、迁移,抑制其凋亡,并诱导其向神经元、星形胶质细胞定向分化。

Objective To observe the effects of astragaloside IV(AST IV)combined with Panax Notoginseng saponins(PNS)on proliferation,apoptosis,migration and neuronal differentiation of oxygen glucosedeprivation/reoxygenation model rat bone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were isolated,cultured,amplified and purified by the whole bone marrow adherent method.The positive expression rates of BMSCs surface markers,CD29,CD90,CD34,and CD45 were detected by flow cytometry.The third generation of BMSCs was pretreated with AST IV and PNS doses of high(100μmol/L+60μmol/L),medium(50μmol/L+30μmol/L),and low(25μmol/L+15μmol/L)for 24 h.The model of ischemia-reperfusion injury was established by OGD/R.Meanwhile,the normal group(BMSCs were cultured normally)and the model group(OGD/R was used to establish an ischemia reperfusion injury model)were established.The cell increment rate was detected by CCK-8 assay.Cell apoptosis was detected by flow cytometry.Transwell assay was used to detect the migration of BMSCs.The condition of BMSCs differentiation into neurons and astrocytes was observed by Nestin/NSE and Nestin/GFAP immunofluorescence double labeling.Results BMSCs were successfully cultured and separated,and the positive rates of CD29 and CD90 detected by flow cytometry were 94.23%and 94.69%,while the positive rates of CD34 and CD45 were 5.76%and 5.31%.Compared with the normal group,the survival rate of the model group was reduced significantly and the apoptosis rate was increased significantly(P<0.05).Compared with the model group,the combination of different doses of AST IV and PNS could promote the proliferation of BMSCs(P<0.05,0.01)and inhibit the apoptosis(P<0.05,0.01).Compared with the normal group,the model group and the AST IV and PNS group at different doses could promote the migration of BMSCs(P<0.05).Compared with the model group,the number of migrated cells in the AST IV and PNS groups at different doses was increased significantly(P<0.05).Compared with the normal group,the model group and the AST IV and PNS groups at different doses could promote the differentiation of BMSCs into neurons and astrocytes(P<0.01).Compared with the model group,the positive expression rates of Nestin/NSE and Nestin/GFAP in the AST IV and PNS groups at different doses were increased significantly(P<0.01).Conclusion AST IV combined with PNS can promote the proliferation and migration of BMSCs of ischemia-reperfusion model in vitro,inhibit the apoptosis,and induce their directional differentiation into neurons and astrocytes.