摘要
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 the lentiviral overexpression vector of ornithine decarboxylase（ODC） and investigate its role in oxidative stress-induced apoptosis in H9C2 cells.Methods The lentiviral vector（pHIV） was constructed by inserting the lentiviral vectors with the ODC gene fragment.The recombination lentiviral particles were produced by the packaging 293T cell,the culture supernatant was harvested and the titration of them was determined by the limiting dilution.H9C2 cells were infected with recombinant lentivirus overexpressing ODC,after 72 hours.The expression level of ODC in cells was examined by the RT-PCR.The effects of ODC overexpression on oxidative stress-induced apoptosis in H9C2 cell were investigated by CCK-8 assay and Hoechst33258 staining.Results The transduction efficiency of H9C2 cell line with pHIV-ODC lentivirus was 95%.72 h after virus infection,ODC mRNA levels were higher than those of negative control group.Compare with H2O2-induced apoptotic cells,cell viability and the amount of apoptotic cells in the ODC overexpressed H9C2 cells was significantly decreased（P0.001）.Conclusion ODC can be overexpressed in H9C2 cells by recombinant lentivirus infection,which leads to a significant inhibition of oxidative stress-induced apoptosis in these cells.
出处
《热带医学杂志》
CAS
2012年第5期510-513,522,F0003,共6页
Journal of Tropical Medicine
基金
国家自然科学基金(30973850
81173459)
广东省科技计划项目(2010B060500009)
