整合素连接激酶增加缺血环境下骨髓间充质干细胞生存率和血管内皮生长因子分泌

Integrin-linked kinase regulates bone mesenchymal stem cells survival and vascular endothelial growth factor secrection partially through protein kinase B and mammalian target of rapamycin signaling pathways

目的 观察整合素连接激酶（ILK）对缺血环境下骨髓间充质干细胞（BMSCs）生存率及血管内皮生长因子（VEGF）分泌的影响并探讨其机制.方法 取大鼠骨髓体外分离扩增培养BMSCs,分别用ILK、ILK-小干扰RNA （siRNA）及对照绿色荧光蛋白（GFP）腺病毒转染BMSCs,无氧无血清条件下培养8h;流式细胞仪检测不同条件下BMSCs的凋亡率,酶联免疫吸附试验（ELISA）法检测不同条件下BMSCs的VEGF分泌水平,Western blot检测蛋白激酶B（Akt）、哺乳动物雷帕霉素靶蛋白（mTOR）磷酸化水平的变化.结果 ILK-BMSCs组ILK的表达显著高于GFP-BMSCs和ILK-siRNA-BMSCs组（P＜0.05）.无氧无血清条件下培养BMSCs 8 h,与BMSCs和GFP-BMSCs组比较,ILK过表达显著增加BMSCs存活率及VEGF表达,且Akt和mTOR的磷酸化水平显著增加（P＜0.05）.而ILK基因沉默则显著减少BMSCs存活率及VEGF表达（P＜0.05）,Akt和mTOR的磷酸化水平显著降低（P＜0.05）.结论 ILK激活其下游信号分子Akt和mTOR,增加缺血环境下BMSCs生存率及VEGF表达.

Objective To investigate the effects of integrin-linked kinase （ILK） gene transfection on bone mesenchymal stem cells （BMSCs） survival and vascular endothelial growth factor （VEGF） secrection under the conditions of serum-free and hypoxia.Methods BMSCs were acquired from the bone marrow of adult rats.The cells were isolated,purified,cultured,and transfeeted with Adv-ILK,Adv-ILK-siRNA,and Adv-green fluorescent protein （GFP） respectively.Cell apoptosis was detected by facial action coding system （FACS）.The concentrations of VEGF in the culture supernatant were measured by enzyme linked immunosorbent assay （ELISA）.The ILK protein level and phospho-protein kinase B （Akt）,Akt,phosphormammalian target of rapamycin （mTOR） were assayed by Western blotting.Results ILK-BMSCs exhibited strong expression of ILK compared with ILK-siRNA-BMSCs and GFP-BMSCs （P 〈 0.05）.Under the conditions of serum-free and hypoxia,upregulation of ILK expression increased the phosphorylation of Akt and mTOR,resulting in markedly enchanced BMSCs survival and VEGF expression （P 〈 0.05） ; while significant inhibition of survival and VEGF expression was detected in BMSCs with ILK kinase inactivation,which was associated with a reduction of phosphorylation of Akt and mTOR （P 〈 0.05）.Conclusion ILK improves the BMSCs survival and VEGF secrection under the conditions of serum-free and hypoxia.