心肌细胞缺氧复氧损伤时Mipu1启动子活性、Mipu1 mRNA表达变化及意义

Changes of Mipu1 promoter activity and mRNA expression during cardiomyocytes hypoxia/reoxygenation injury and its significance

目的探讨心肌细胞缺氧复氧损伤时心肌缺血预适应表达上调蛋白1(Mipu1)启动子活性的变化及其作用。方法按本课题组的常规方法复制心肌细胞缺氧复氧模型,先予缺氧(95%N2-5%CO2,无血清低糖DMEM)处理6 h,再分别复氧(95%空气-5%CO2,10%FBS-高糖DMEM)6、12、24 h。采用MTT法检测心肌细胞活性,比色法测定心肌细胞LDH活性,双荧光素酶报告基因技术检测启动子PGL3-Mipu1活性,实时定量PCR法检测Mipu1mRNA表达。结果缺氧6 h复氧(6、12、24 h)后心肌细胞活性减低,LDH释放增加,启动子PGL3-Mipu1活性增加,Mipu1 mRNA表达上调,且在缺氧6 h复氧12 h时变化最明显。结论 Mipu1可能参与了心肌细胞的缺氧复氧损伤,其可能通过调控凋亡相关基因发挥作用。

Objective To investigate the changes and roles of Mipul promoter activity during cardiomyocytes hypoxia/ reoxygenation injury. Methods The cardiomyocytes H9c2 injury model was induced by hypoxia/reoxygenation, H9c2 cells were transferred to a hypoxic incubator that contained 95% N2-5% CO2 for 6 hs of hypoxia, and then were transferred to a normoxic incubator （95% air-5% CO2 ） for 6, 12 and 24 hs. The activity of H9c2 cells was detected by MTF method. Serum LDH activity was measured by chromatometry. PGL3-Mipul promoter activity was dectected by luciferase reporter assay. Mipul mRNA expression was tested by real-time PCR. Results After the hypoxia/reoxygenation , the cell survival rates decreased, LDH activity and Mipul promoter activity increased, Mipul mRNA expression upregulated, especially at hypoxia 6 h/12 h of reoxygenation. Conclusions Mipul may participate in the cardiomyocytes injury induced by hypoxia/ reoxygenation, which maybe partly due to its regulation of apoptosis-related gene expression.