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目的构建过表达鸟氨酸脱羧酶(ODC)基因慢病毒载体,并研究其对氧化应激诱导大鼠H9C2心肌细胞凋亡的影响。方法构建大鼠ODC基因pHIV-ODC过表达质粒,与包装质粒psPAX2、pMD2G共转染293FT细胞,检测其侵染效率;包装慢病毒并侵染H9C2心肌细胞;72 h后观察其侵染效率,RT-PCR法检测细胞ODC mRNA的表达,利用CCK-8、Hochest33258染色检测ODC对H2O2诱导H9C2心肌细胞凋亡的影响。结果酶切及测序鉴定ODC序列正确插入慢病毒过表达载体。侵染H9C2细胞72 h后ODC mRNA水平较阴性慢病毒感染组显著升高。过表达ODC能抑制H2O2诱导的细胞活力下降,减轻H2O2诱导的心肌细胞凋亡,与H2O2组比较差异均有统计学意义(P<0.001)。结论成功构建的过表达ODC慢病毒,上调H9C2细胞中ODC的表达,过表达ODC能显著抑制氧化应激诱导的H9C2心肌细胞凋亡。
Objective To construct lentiviral vector overexpressing ornithine decarboxylase (ODC) gene and study its effect on oxidative stress-induced apoptosis of H9C2 cardiomyocytes. Methods The ODN gene ODN-ODC overexpression plasmid was constructed and cotransfected into 293FT cells with packaging plasmid psPAX2 and pMD2G to detect the infection efficiency. The lentivirus was infected and infected with H9C2 cardiomyocytes. The infection efficiency, The expression of ODC mRNA was detected by RT-PCR. The effect of ODC on H2O2-induced H9C2 cardiomyocyte apoptosis was examined by CCK-8 and Hochest33258 staining. Results Enzyme digestion and DNA sequencing confirmed that the ODC sequence was correctly inserted into lentivirus overexpression vector. The ODC mRNA level in H9C2 cells infected 72 h after infection was significantly higher than that in the negative lentivirus group. Overexpression of ODC could inhibit H2O2-induced decrease of cell viability and decrease of H2O2-induced cardiomyocyte apoptosis compared with H2O2 group (P <0.001). Conclusions ODC lentivirus overexpressed and up-regulated the ODC expression in H9C2 cells. Overexpression of ODC significantly inhibited oxidative stress-induced H9C2 cardiomyocyte apoptosis.