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目的观察Bcl-2家族蛋白在Ad.mda-7介导HepG2细胞凋亡中的变化,初步探讨Ad.mda-7介导肝癌细胞的凋亡机制。方法将携带人MDA-7/IL-24基因的腺病毒转染HepG2和L02,通过四甲基偶氮哩蓝染色法(MTT)及Hoechst染色观察MDA-7/IL-24对肝癌细胞HepG2和正常肝胚细胞L02的生长抑制和杀伤作用。Annexin V和PI双染后流式细胞仪检测细胞的凋亡。蛋白免疫印迹法(Western blot)检测Bcl-2家族蛋白的变化。结果Ad.mda-7能明显抑制肝癌细胞HepG2的生长,但对正常肝胚细胞L02没有明显抑制增殖作用。Hoechst染色和流式细胞仪提示Ad.mda-7能明显诱导肝癌细胞HepG2细胞的凋亡,但对正常肝胚细胞L02没有明显毒副作用。Western blot说明抑制凋亡的蛋白Bcl-2和Bcl-xl的表达在HepG2明显下降,在L02中则没有变化;而促凋亡蛋白Bax在HepG2和L02都升高。结论Ad.mda-7转染HepG2后,促凋亡(Bax)与抑凋亡蛋白(Bcl-2和Bcl-xl)的比率发生改变,进而发生凋亡。
Objective To observe the changes of Bcl-2 family protein in HepG2 cells induced by Ad.mda-7 and to explore the mechanism of apoptosis induced by Ad.mda-7. Methods Adenovirus carrying human MDA-7 / IL-24 gene was transfected into HepG2 and L02 cells. The effects of MDA-7 / IL-24 on HepG2 and Hp were observed by MTT assay and Hoechst staining Growth inhibition and cytotoxicity of normal hepatocyte L02. Annexin V and PI double staining after flow cytometry cell apoptosis. Western Blot was used to detect the changes of Bcl-2 family proteins. Results Ad.mda-7 can significantly inhibit the growth of HepG2 hepatoma cells, but did not inhibit the proliferation of normal hepatocytes L02. Hoechst staining and flow cytometry suggested that Ad.mda-7 could obviously induce apoptosis of HepG2 cells, but had no obvious toxic and side effects on normal liver cells. Western blot analysis showed that the expression of Bcl-2 and Bcl-xl in HepG2 cells decreased significantly in L02 and Bax in HepG2 and L02 cells. Conclusions After HepG2 was transfected with Ad.mda-7, the ratio of Bax to Bcl-2 and Bcl-xl was changed, and then apoptosis occurred.