人双突变型低氧诱导因子α促进骨髓间充质干细胞向心肌细胞分化的实验研究

Effects of a recombinant adenovirus expressing human hypoxia-inducible factor 1α double-mutant on the in vitro differentiation of bone marrow mesenchymal stem ceils to cardiomyocytes

目的 探讨人双突变型低氧诱导因子1α（HIF-1α）基因（HIF-1α-402-564）对与心肌细胞共培养的骨髓间充质干细胞（MSC）向心肌细胞分化的影响.方法 实验分为4组：HIF-1α组（MSC＋心肌细胞＋Ad-HIF-1α）、LacZ组（MSC＋心肌细胞＋Ad-Lacz）、Sham组[MSC＋心肌细胞＋胎牛血清（PBS）]、MSC＋HIF-1α组（MSC＋Ad-HIF-1α）,前三组中MSC与心肌细胞按1：2比例共同培养,各组细胞培养24 h后分别感染不同病毒（MOI=100）或加入PBS液.病毒感染后7 d,免疫细胞化学分析共培养的MSC表达心肌细胞特异性标志物心肌肌钙蛋白T（cTnT）的情况,逆转录聚合酶链式反应（RT-PCR）分析各组细胞HIF-1α、转化生长因子β1（TGF-β1）、Smad4、NKx2.5、GATA结合蛋白4（GATA-4）的mRNA表达量.结果 HIF-1α组MSc心肌细胞分化率为（32.68±6.52）%,显著高于LacZ组[（8.28±0.09）%]和Sham组[（10.25±2.20）%],P均〈0.05,MSC±HIF-1α组仅为（0.32±0.05）%.LacZ组和Sham组间差异无统计学意义.MSC＋HIF-1α组与Sham组比较,差异有统计学意义（P〈0.05）.HIF-1α组TGFβ1及Smad4 mRNA表达水平明显高于其余各组,P均〈0.05.HIF-1α组NKx2.5和GATA-4 mRNA表达水平明显高于Sham组,P均〈0.05.结论 HIF-1α能够促进与心肌细胞共培养的MSC向心肌细胞分化,TGF-β1/Smad4信号通路参与了该过程.

Objective To observe the effects of mutant hypoxia-inducible factor-1α （ HIF-1α） adenovirus （ Adeno-HIF-1α-Ala402-Ala564 ） on cardiomyocytes （ CMCs ） differentiation from the mesenchymal stem cells （ MSCs） co-cultured with CMCs. Methods Following groups were studied： HIF-1α group （ MSCs ＋ CMCs ＋ Ad-HIF-1α）, LacZ group （ MSCs ＋ CMCs ＋ Ad-LacZ） , Sham group （MSCs ＋ CMCs ＋ PBS） and MSC ＋ HIF-la Group （ MSCs ＋ Ad-HIF-1α）. MSCs were co-cultured with myocardial cells in proportion of MSCs： CMCs 1：2, after 24 hours, cells were infect with virus （MOI = 100） or treated with PBS, cardiac troponin （cTnT） expression in MSCs was detected 7 days post infection by immunochemical analysis, mRNA expression of HIF-la, TGF-B,, Smad4, NKx2.5, GATA-4 was also detected by RT-PCR. Results HIF-la increased MSCs differentiation to myocardial cells （differentiation rate 32. 68%±6. 52% vs. 8. 28% ± 0.09% in the LacZ group and 10. 25 % ±2. 20% in the Sham group and 0.32% ±0.05% in the MSC group （all P〈 0.05 vs. HIF-la group）. mRNA expression of HIF, TGF-β1, Smad4, NKx2. 5 and GATA-4 was also significantly upregulated in HIF-1α group all P 〈0. 05 vs. Sham group）. Conclusion HIF-1α promoted MSCs, co-cultured with myocardial cells, differentiating to cardiomyocytes via upregulating TGF-β1/Smad4 signaling pathway.