人骨髓间充质干细胞定向血管内皮细胞诱导分化前后eNOS表达/活性及其代谢产物的差异

摘要

目的 探讨人骨髓间充质干细胞(hBMSCs)定向血管内皮细胞诱导分化前后内皮型一氧化氮合酶(eNOS)基因表达、活性及其代谢产物的差异.方法 建立hBMSCs定向血管内皮细胞诱导分化系统.应用倒置显微镜观察细胞形态的改变,Transwell实验检测细胞的迁移能力,细胞免疫荧光及Western blot检测eNOS的蛋白表达,ELISA法检测eNOS的活性,硝酸还原法检测细胞培养上清液NO含量.结果 与未分化组相比,分化后的细胞形态发生明显改变;细胞的迁移能力增强238.10％ (73.000±7.002 vs.21.000±4.359,P＜0.05);eNOS蛋白的表达增加114.72％(0.423±0.011 vs.0.197±0.079,P＜0.05);eNOS的活性增强157.49％(4.967±0.073 vs.1.929±0.103,P＜0.05);NO合成与释放增加155.67％(184.909±1.853 vs.72.323±0.426,P＜0.05).结论 hBMSCs定向内皮细胞诱导分化后eNOS基因表达增加、活性增强,其代谢产物NO的合成与释放增加.本结果可能为干细胞在心血管疾病等防治提供依据.%Objective To investigate the differences in eNOS gene expression,activity and its metabolites before and after human bone marrow mesenchymal stem cells (hBMSCs) are induced into vascular endothelial cells.Methods hBMSCs were induced into vascular endothelial cells.The morphological changes of the cells were observed under inverted microscope.Transwell assay was used to detect the cells' migration ability.The protein expression of eNOS was detected by immunofluorescence and Western blot.The activity of eNOS was detected by ELISA and the content of NO in cell culture supernatant was determined by nitrate reduction method.Results Compared with those in the undifferentiated group,the morphological changes of the differentiated cells were obvious.Cell migration ability increased by 238.10％ (73.000±7.002 vs.21.000±4.359,P＜0.05).The expression of eNOS protein increased by 114.72％ (0.423±0.011 vs.0.197±0.079,P＜0.05).The activity of eNOS was enhanced by 157.49％ (4.967±0.073 vs.1.929±±0.103,P＜0.05).The synthesis and release of NO increased by 155.67％ (184.909±1.853 vs.72.323±0.426,P＜0.05).Conclusion After hBMSCs are induced into endothelial cells,the expression of eNOS gene increases,their activities increase,synthesis and release of the metabolite NO increase.It may provide a basis for prevention and treatment of cardiovascular diseases with stem cells.