谷胱甘肽过氧化物酶2干涉慢病毒系统构建及对人肝癌细胞凋亡的影响

Construction a lentiviral vector for RNA interference of glutathione peroxidase 2 gene and its effect on HepG2 cell apoptosis

目的构建人谷胱甘肽过氧化物酶2(Glutathione peroxidase 2,GPX2)慢病毒干涉载体,获得稳定下调GPX2的高滴度慢病毒,检测敲低GPX2对细胞凋亡的影响。方法通过筛选具有显著干涉效果的si RNA序列,构建p Sico R-GPX2慢病毒干涉载体,包装病毒并感染人肝癌Hep G2细胞,分别用Western-blot和RT-PCR方法检测GPX2表达情况,应用流式细胞术分析敲低GPX2对人肝癌细胞凋亡的影响。结果成功构建p Sico R-GPX2干涉慢病毒载体并获得高滴度慢病毒颗粒,GPX2蛋白表达水平和RNA表达水平均有显著下调,且人肝癌细胞感染GPX2干涉慢病毒后对凋亡更加敏感,其原因可能是促凋亡蛋白Bax的活化和抗凋亡蛋白Bcl-2活性的抑制。结论成功构建人GPX2基因干涉慢病毒,为肝癌靶标治疗提供新的线索,为后续GPX2功能研究奠定基础。

Objective To construct a RNA interference lentiviral vector for human glutathione peroxidase 2（GPX2） gene and observe the effect of GPX2 knockdown on cell apoptosis. Methods The sequence of the small interfering RNA（si RNA） for GPX2 interference was inserted into the p Sico R vector. Hep G2 cells were infected by the packaged si-GPX2 lentivirus and the expression of GPX2 in the infected cells was detected by both RT- PCR and Western blotting. Changes of cell apoptosis following the infection were analyzed by flow cytometry. Results The lentiviral particles p Sico R- GPX2 were successfully packaged. The expression of GPX2 in the infected cells was obviously down-regulated at both RNA and protein levels. GPX2 knockdown caused increased apoptosis rate, increased Bax expression and lowered Bcl- 2 expression in Hep G2 cells.Conclusion We have successfully constructed the lentiviral vector for RNA interference of human GPX2 gene.