缺氧条件下大鼠脑微血管内皮细胞对共培养的人间充质干细胞分化的影响

Effect of brain microvascular endothelial cells on differentiation of co-cultured mesenchymal stem cells under hypoxic conditions

目的探讨缺氧条件下大鼠脑微血管内皮细胞(brain microvascular endothelial cells,BMECs)对人间充质干细胞(mesenchymal stem cells,MSCs)分化的影响。方法分离、培养并鉴定人骨髓MSCs和大鼠BMECs,在正常和缺氧两种条件下,分别以间接和直接共培养两种方式对MSCs进行诱导分化,用流式细胞术和免疫荧光细胞化学法观察和分析诱导后MSCs的胎肝激酶-1(fetal liver kinase-1,Flk-1)和血管性血友病因子(von Willebrand factor,vWF)的表达。结果正常间接共培养未能诱导MSCs表达vWF和Flk-1蛋白,缺氧间接共培养和正常条件下的直接共培养均能诱导MSCs开始表达Flk-1蛋白,而vWF染色仍为阴性,缺氧条件下直接共培养表达Flk-1蛋白的MSCs比正常条件增多,而且部分Flk-1阳性细胞开始同时表达vWF蛋白。结论BMECs能够通过细胞直接接触诱导共培养的MSCs开始向内皮分化,缺氧条件下的直接共培养BMECs能诱导更多的MSCs更彻底地向内皮分化。

Objective To investigate the effect of brain microvascular endothelial cells （BMECs） on mesenchymal stem cells （MSCs） expressing fetal liver kinase-1 （Flk-1） and yon Willebrand factor （vWF） under normal and hypoxic conditions. Methods Human bone marrow MSCs and rat BMECs were isolated, cultured and identified. We used direct and indirect co-culture to induce the differentiation of MSCs. The expression of Flk-1 and vWF after induction was observed and analyzed by flow cytometry and immunofluorescence staining. Results Undifferentiated MSCs did not express Flk-1 or vWF. In indirect co-culture group, indirect co-culture under hypoxic conditions induced （7.58 ± 0.58） % （n = 6, P = 0. 034） MSCs to express Flk-1 but not vWF, while in normoxic indirect co-culture, MSCs expressed neither. In direct co-culture group, after 5-day direct co-culture with BMECs under normoxia or hypoxia, MSCs expressing Flk-1 accounted for （13.76 ± 1.67） % （n = 6, P 〈 0.01 ） and （23.64 ± 2.50） % （ n = 6, P 〈 0.01 ） of the total co-cultured mixed cells, and the vWF staining was still negative in the normoxic co-culture system, while some Flk-1 positive cells began to co-express vWF simultaneously in the hypoxic co-culture system. Conclusion BMECs induce MSCs to differentiate into endothelial cells through direct cell contact. By direct co-culture in hypoxic conditions, BMECs induce more MSCs to differentiate into endothelial cells.