过表达HSP20的慢病毒载体对H_2O_2诱导的大鼠心肌细胞凋亡的影响

Effect of the lentiviral vector overexpressing HSP20 on the myocardial apoptosis induced by H_2O_2

目的构建过表达大鼠热休克蛋白20(HSP20)基因慢病毒载体,探讨其对H2O2诱导的大鼠H9C2心肌细胞凋亡的影响。方法构建大鼠HSP20基因pHIV-HSP20过表达质粒慢病毒,与包装质粒psPAX2、pMD2G共转染293FT细胞,检测其转染效率;包装慢病毒并转染H9C2心肌细胞;72 h后观察其转染效率,RT-PCR法检测细胞HSP20 mRNA表达,CCK-8、Hochest33258染色法检测H2O2诱导后H9C2心肌细胞凋亡情况。结果成功构建了HSP20慢病毒过表达载体,经293FT细胞包装后,其对H9C2细胞72 h的转染效率为95%;与慢性病毒组比较,H9C2细胞转染72 h后HSP20 mRNA水平显著升高,细胞活力下降,心肌细胞凋亡率明显降低(P均<0.01)。结论过表达HSP20能显著抑制H2O2诱导的H9C2心肌细胞凋亡。

Objective To construct the lentiviral vector overexpressing heat shock protein 20 （ HSP20）, and to investigate its effect on the apoptosis of myocardial H9C2 Cells induced by H2O2. Methods The lentiviral vector （pHIV） was constructed by inserting the lentiviral vectors with the HSP20 gene fragment. The recombination lentiviral particles were produced by the packaging 293T cell, then the transduction efficiency was detected. H9C2 cells were infected with recom- binant lentivirus overexpressing HSP20, and the expression level of HSP20 in cells was examined by the RT-PCR 72 hs lat- er. The H2O2 induced apoptosis in H9C2 cells was investigated by CCK-8 assay and Hoechst33258 staining method. Results pHIV-HSP20 lentivirus was constructed successfully, the transduction efficiency of H9C2 cell line was 95%, 72 hs after virus infection; compared with the lentiviral group, the HSP20 mRNA level of H9C2 cell increased significantly, cell viability and the amount of apoptotic cells decreased significantly, 72 hs after virus infection（ all P 〈 0.01 ）. Conclusion Overexpression of HSP20 can significantly inhibit the myocardial apoptosis induced by H2O2.