低氧激活自噬促进人间充质干细胞成骨分化的研究

Study of Hypoxia Induced Autophagy on Human Mesenchymal Stem Cells Differentiation

目的探讨低氧对人间充质干细胞(human mesenchymal stem cells,h MSCs)成骨分化的效应及分子机制。方法在正常氧压(20%O_2)和低氧(5%O_2)条件下培养hMSCs;采用成骨分化诱导液(BDM)诱导h MSCs成骨分化;利用茜素红染色法考察细胞钙沉积情况;通过Western blotting检测细胞自噬相关蛋白的表达水平以及MAPK、PI3K-AKT-mTOR信号通路的激活情况。结果低氧条件下,h MSCs钙沉积较常氧条件增强;BDM诱导h MSCs成骨分化过程伴随着LC3表达增加,抑制细胞自噬可以明显减弱低氧促进的细胞钙沉积;低氧促进的细胞发生钙沉积伴随着MAPK以及PI3K-AKT-m TOR信号通路的失活;抑制MAPK以及PI3K-AKT-m TOR信号通路有利于细胞自噬发生,进而促进h MSCs钙沉积。结论低氧下可以通过激活细胞自噬来促进h MSCs成骨分化,抑制MAPK以及PI3K-AKT-m TOR信号通路可以进一步促进细胞自噬的发生,从而协同促进h MSCs成骨分化。

OBJECTIVE To explore the effects and molecular mechanisms of hypoxia on h MSCs differentiation. METHODS h MSCs were cultured under normoxic or hypoxic conditions; h MSCs differentiation were obtained by treated with BDM（bone differentiation medium）; Calcium deposit was assessed by Alizard Red S dye; The protein levels were analyzed by western blotting. RESULTS Hypoxia can significantly promoted calcium deposit in h MSCs; The expression of LC3 was increased in h MSCs, hypoxia induced calcium deposit was inhibited by autophagy inhibitor 3MA; Hypoxia induced h MSCs calcium deposit was accompanied by inactivation of MAPK and PI3K-AKT-m TOR pathway, inhibition of these pathway could mediate h MSCs calcium deposit by promoted autophagy. CONCLUSION Hypoxia can significantly triger h MSCs differentiation by induced autophagy, inhibition of MAPK and PI3K-AKT-m TOR pathway could contribute to autophagy which could mediate h MSCs differentiation.