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目的探讨突变型低氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)基因对体外诱导环境人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)分化成心肌样细胞(cardiomyocyte-like)过程中的影响。方法重组腺病毒Ad-HIF-1αnature、Ad-HIF-1α564、Ad-HIF-1α564/402和Ad-HIF-1α564/803以最佳转染复数转染体外5-氮胞苷(5-azacytidine,5-aza)诱导的hMSCs,诱导培养后第4周,提取细胞浆蛋白,一步法酶联免疫分析检测心肌肌钙蛋白I(cTnI)含量;Western blot测定HIF-1α及VEGF蛋白表达水平。结果5-aza诱导hMSCs第4周cTnI值:Ad-HIF-1αnature(0.426±0.001)、Ad-HIF-1α564(0.424±0.002)、Ad-HIF-1α564/402(0.428±0.005)、Ad-HIF-1α564/803(0.441±0.003)、空白组(0.411±0.010)、Ad-LacZ(0.410±0.016),各实验组有显著性差异(P<0.05);Ad-HIF-1α564/803组cTnI表达高于Ad-HIF-1αnature、Ad-HIF-1α564、Ad-HIF-1α564/402三组(P<0.05)。Western blot结果显示Ad-HIF-1αnature、Ad-HIF-1α564、Ad-HIF-1α564/402和Ad-HIF-1α564/803四组HIF-1α及VEGF蛋白表达水平均较Ad-LacZ组高,有显著性差异(P=0.000),但Ad-HIF-1α564、Ad-HIF-1α564/402和Ad-HIF-1α564/803与Ad-HIF-1αnature相比,各组间两种蛋白表达差异均无显著性(P>0.05)。结论野生型HIF-1α基因及突变型能促进5-aza诱导的hMSCs向心肌样细胞转化,为HIF-1α及hMSCs在心肌再生中的联合应用奠定了实验基础。
Objective To investigate the effects of hypoxia inducible factor-1α (HIF-1α) gene on the differentiation of human mesenchymal stem cells (hMSCs) into cardiomyocyte-like cells In the impact. Methods Recombinant adenovirus Ad-HIF-1αnature, Ad-HIF-1α564, Ad-HIF-1α564 / 402 and Ad-HIF-1α564 / 803 were transfected in vitro with 5-azacytidine 5-aza) induced hMSCs. At 4 weeks after induction, cytosolic protein was extracted and the content of cardiac troponin I (cTnI) was detected by one-step enzyme-linked immunosorbent assay. The protein expression of HIF-1α and VEGF was detected by Western blot. Results The cTnI of Ad-HIF-1αnature (0.426 ± 0.001), Ad-HIF-1α564 (0.424 ± 0.002), Ad-HIF-1α564 / 402 1α564 / 803 (0.441 ± 0.003), blank group (0.411 ± 0.010) and Ad-LacZ (0.410 ± 0.016), respectively. There was significant difference between the experimental groups (P <0.05); cTnI expression in Ad-HIF-1α564 / 803 group Higher than that of Ad-HIF-1αnature, Ad-HIF-1α564 and Ad-HIF-1α564 / 402 (P <0.05). Western blot results showed that the expression of HIF-1α and VEGF protein in Ad-HIF-1αnature, Ad-HIF-1α564, Ad-HIF-1α564 / 402 and Ad-HIF-1α564 / (P = 0.000). However, compared with Ad-HIF-1αnature Ad-HIF-1α564, Ad-HIF-1α564 / 402 and Ad-HIF-1α564 / 803, there was no difference between the two groups Significance (P> 0.05). Conclusions Wild-type HIF-1α gene and mutant can promote 5-aza-induced hMSCs transdifferentiation into cardiomyocyte-like cells, which provides an experimental basis for the combined application of HIF-1α and hMSCs in myocardial regeneration.