hHO-1联合GATA-4修饰大鼠骨髓间充质干细胞抗凋亡及向心肌分化的实验研究

摘要

Objectives To explore if the rat bone marrow mesenchymal stem cells (BMSCs) modified by human heme oxygenase 1 (hHO-1) gene combined with GATA-4 gene may promote the ability of anti-apoptosis and myocardial transdifferentiation in vitro in hypoxia ischemic environment.Methods The rat BMSCs were isolated and cultured by whole bone marrow adherence and identified in vitro,and then were transfected with recombinant adenovirus;Western blotting was used to determinate the optimal time of gene expression;the genetically modified BMSCs were taken to hypoxia serum-free conditions simulating ischemia hypoxia microenvironment in vivo;CCK-8 kit and trypan blue staining were performed to detect the 12,24,48 and 72h survival rates in hypoxia ischemia respectively;flow cytometry was used to detect the apoptosis of BMSCs in hypoxia ischemia for 24h.The cardiomyocyte-specific cardiac troponin Ⅰ (cTnI) was detected by Western blotting and cellular immunofluorescence.Results The 12,24,48 and 72h survival rates were higher in hHO-1+GATA-4 group cultured in ischemia and hypoxia condition than in hHO-1 group (P＜0.05) and GATA-4 group (P＜0.05).After 24h cultivation in ischemia hypoxia condition,the apoptotic rates were lower in hHO-1+GATA-4 group than in hHO-1 group (P＜0.05) and GATA-4 group (P＜0.05).No significant difference existed in cTnI expressions between GATA-4 group and hHO-1+GATA-4 group.Conclusion Compared with transfection of hHO-1 or GATA-4 single gene,hHO-1 combined with GATA-4 transduction can significantly increase the survival rate of BMSCs in hypoxia ischemic condition,but myocardial transdifferentiation does not increase significantly.%目的 探讨经人血红素加氧酶1 (hHO-1)基因和GATA-4基因联合修饰的大鼠骨髓间充质干细胞(BMSCs)在缺血缺氧环境中抗凋亡及体外分化为心肌细胞样细胞的能力是否优于hHO-1或GATA-4单基因修饰的BMSCs.方法 采用全骨髓贴壁法分离、培养BMSCs并进行鉴定,重组腺病毒转染BMSCs,Western blotting法确定基因表达的最佳时间;以缺氧无血清条件模拟体内缺血缺氧微环境培养基因修饰的BMSCs,采用CCK-8试剂盒和台盼蓝染色法分别检测缺血缺氧培养12、24、48、72h细胞存活率,流式细胞术检测缺血缺氧培养24h细胞凋亡情况,Western blotting及细胞免疫荧光法检测特异性心肌肌钙蛋白Ⅰ(cTnI)表达情况.结果 缺血缺氧培养12、24、48、72h的hHO-1 +GATA-4组细胞生存率均高于hHO-1组(P＜0.05)和GATA-4组(P＜0.05).缺血缺氧培养24h的hHO-1 +GATA-4组细胞凋亡率低于hHO-1组(P＜0.05)和GATA-4组(P＜0.05);GATA-4组与hHO-1 +GATA-组的cTnI表达差异无统计学意义.结论 hHO-1与GATA-4联合修饰可明显增强缺血缺氧BMSCs的存活;与GATA-4单基因修饰相比,联合修饰并没有增强BMSCs向心肌细胞分化的能力.