HIF-1α/apelin/APJ通路在缺氧预处理促进心肌干细胞增殖和向心肌样细胞分化中的作用

摘要

BACKGROUND: Our previous studies demonstrated that cardiac stem cells (CSCs) transplantation could improve cardiac function in rats with myocardial infarction (MI). However, the overall survival and cardiac differentiation of CSCs were low. OBJECTIVE: To investigate the effect of hypoxia preconditioning on CSCs proliferation and cardiogenic differentiation and the role of hypoxia induced factor-1alpha (HIF-1α)/apelin/putative receptor protein related to the angiotensin receptor AT1 (APJ) pathway in the procedure. METHODS: Cells cultured in vitro experienced exposure to hypoxia (1% O2) for 24 hours. Cardiogenic differentiation was induced by using 5-azacytidine for another 24 hours. Then, cells were cultured in normal condition for 2 weeks. Normoxia (20% O2) was used as a negative control during the whole process. Cell proliferation was detected using MTS method and expressions of HIF-1α, apelin, cTnT and APJ were detected using western blot assay after 24 hours of preconditioning and 2 weeks after the induction of differentiation; the percentage of cTnT-positive cardiomyocyte-like cells was observed by immunofluorescence staining. RESULTS AND CONCLUSION: Compared with the normoxia group, the hypoxia group presented a higher proliferation rate and a higher absorbance value at 490 nm (P < 0.01); the protein expressions of HIF-1α, apelin and APJ were all enhanced after hypoxia exposure for 24 hours and 2 weeks after the induction of differentiation (P < 0.01); the percentage of cTnT-positive cells was greatly increased in the hypoxia group (P < 0.01), and the expression of cTnT was also significantly intensified (P < 0.01). To conclude, hypoxia preconditioning could promote the proliferation and cardiogenic differentiation of CSCs, and the activation of HIF-1α/Apelin/APJ pathway might be involved in this process.%背景:课题组前期研究显示心肌干细胞移植能够改善大鼠心肌梗死后心功能,但心肌干细胞在局部心肌梗死组织中的生存及心肌分化效率低下.目的:体外实验观察缺氧预处理对心肌干细胞增殖和向心肌样细胞分化的影响并探讨HIF-1α/apelin/APJ通路在其中的作用.方法:体外培养的心肌干细胞分为缺氧组(体积分数为1%O2)和常氧组(体积分数为20%O2),培养24 h后,MTS法检测两组细胞的增殖情况,Western blot检测缺氧诱导因子1α、apelin、APJ的表达;两组细胞用5-氮杂胞苷诱导分化24 h,再进行正常培养2周,Western blot检测缺氧诱导因子1α、apelin、APJ以及cTnT的表达;免疫荧光染色观察cTnT阳性的心肌样细胞的比例.结果与结论:①与常氧组比较,缺氧组在培养24 h后的增殖率和A490值均显著增高(P<0.01);②与常氧组比较,缺氧组在培养24 h和诱导分化2周后缺氧诱导因子1α、apelin和APJ的蛋白表达量均明显增高(P<0.01);③与常氧组比较,缺氧组诱导分化2周后cTnT阳性的心肌样细胞的比例显著增加(P<0.01),cTnT蛋白表达量明显增高(P<0.01);④结果表明,缺氧预处理能够促进心肌干细胞的增殖和向心肌样细胞发生分化,此效应可能与缺氧诱导因子1α/apelin/APJ通路的激活相关.