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

发布时间：2018-02-25 21:14

  本文关键词： 间质干细胞 基因 GATA 血红素加氧酶- 心肌分化　出处：《解放军医学杂志》2017年04期 　论文类型：期刊论文

【摘要】：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(P0.05)和GATA-4组(P0.05)。缺血缺氧培养24h的hHO-1+GATA-4组细胞凋亡率低于hHO-1组(P0.05)和GATA-4组(P0.05);GATA-4组与hHO-1+GATA-组的c Tn I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。
[Abstract]:Objective to investigate the ability of rat bone marrow mesenchymal stem cells modified by human heme oxygenase 1hHO-1 gene and GATA-4 gene to inhibit apoptosis and differentiate into cardiomyocyte-like cells in vitro under ischemia and hypoxia environment. Methods the ability of BMSCs modified by human heme oxygenase (HO-1) gene and GATA-4 gene to differentiate into cardiomyocyte-like cells in vitro was better than that of hHO-1 or GATA-4 alone. Methods whole bone marrow adherent method was used to isolate BMSCs. BMSCs was cultured and identified. The optimal time for gene expression was determined by Western blotting method. CCK-8 kit and Trypan blue staining were used to detect the survival rate of cells cultured with anoxia and anoxia for 72 h. Flow cytometry (FCM) was used to detect the apoptosis of hHO-1 GATA-4 cells cultured with ischemia and hypoxia for 24 h and the expression of specific cardiac troponin I cTnI by immunofluorescence assay. Results the cell survival rate of hHO-1 GATA-4 group was higher than that of hHO-1 group at 72 h after ischemia and hypoxia culture. The apoptosis rate of hHO-1 GATA-4 group was lower than that of hHO-1 group (P 0.05) and that of GATA-4 group was not significantly different from that of hHO-1 GATA-4 group and hHO-1 GATA-group. Conclusion hHO-1 combined with GATA-4 modification can significantly enhance the survival of hypoxic ischemic BMSCs. Compared with GATA-4 single gene modification, combined modification did not enhance the ability of BMSCs to differentiate into cardiomyocytes.
【作者单位】： 南方医科大学研究生院;郴州市第一人民医院高通量分子诊断技术国家&地方联合工程实验室;解放军305医院中心实验室;
【基金】：国家自然科学基金(30971235) 军队“十二五”医学科研基金(2013XL010)~~
【分类号】：R542.22
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