莱菔硫烷对人肝癌HepG-2细胞Bcl-2与Bax基因转录及蛋白表达的影响

Effects of Sulforaphane on Gene Transcription and Protein Expression of Bcl-2 and Bax in HepG-2 Cells

目的:研究莱菔硫烷(Sulforaphane,SFN)在体外对HepG-2细胞Bcl-2、Bax基因转录和蛋白表达的影响,探讨SFN诱导人肝癌HepG-2细胞凋亡的机制。方法:不同浓度SFN处理体外培养的HepG-2细胞株48 h,采用SRB法测定SFN对HepG-2细胞增殖的影响;激光共聚焦显微镜观察凋亡细胞形态;流式细胞仪检测SFN作用后的细胞凋亡率;Western blot法和RT-PCR法检测SFN在蛋白和mRNA水平上对Bcl-2和Bax表达的影响。结果:SFN对HepG-2细胞的GI50为9.40μmol/L,TGI为26.68μmol/L;10、20、40μmol/L的SFN作用于HepG-2细胞48 h后,激光共聚焦显微镜下呈现典型的凋亡细胞形态,细胞凋亡率分别为(27.42±0.43)%、(46.53±0.35)%和(58.92±0.48)%(P<0.01);细胞内Bcl-2蛋白和mRNA表达水平均显著降低(P<0.01),而20、40μmol/L的SFN作用后细胞内Bax蛋白和mRNA的表达水平均显著升高(P<0.01),Bcl-2/Bax和Bcl-2 mRNA/Bax mRNA均随SFN剂量的增加显著降低(P<0.01),且变化趋势均呈一定的剂量依赖关系。结论:SFN能抑制HepG-2细胞的增殖,诱导HepG-2细胞凋亡,可在翻译和转录水平下调Bcl-2、上调Bax的表达,这可能是SFN诱导HepG-2细胞凋亡的主要机制之一。

Objective：To investigate the effect of Sulforaphane（SFN） on gene transcription and protein expression of Bcl-2 and Bax in HepG-2 Cells so as to explore its mechanism for inducing apoptosis of HepG-2 cells.Methods：HepG-2 cells were treated with different concentrations of SFN for 48h.The effect of SFN on the rates of cell proliferation was detected with SRB assay,the morphology changes of HepG-2 cells were observed with confocal laser scanning microscope（CLSM）,the apoptosis rate of HepG-2 cells was analyzed with flow Cytometry（FCM）,and the effects of SFN on protein and mRNA expression of Bcl-2 and Bax were measured by RT-PCR and Western blot methods respectively.Results：SFN could inhibit the proliferation of HepG-2 cells in vitro,and the GI50 and TGI of SFN were 9.40 μmol/L and 26.68 μmol/L.After treated with SFN at the dosage of 10,20 and 40 μmol/L for 48 h,cells presented the typical modality of early apoptosis,and the apoptosis rates were（27.42±0.43）%,（46.53±0.35）% and（58.92±0.48）% respectively（P0.01）,and the expression of Bcl-2 protein and mRNA were signifcantly decreased（P0.01）.While,after treated with SFN at the dosage of 20,40 μmol/L for 48 h,the expresstion of Bax and mRNA were increased remarkably（P0.01）,and the ratios Bcl-2/ Bax and Bcl-2 mRNA/ Bax mRNA were decreased signifcantly（P0.01） in dose-dependent manner.Conclusion：SFN can inhibit the proliferation and induce cell apoptosis of HepG-2 cells.The down-regulating expression of Bcl-2 and up-regulating expression of Bax on both mRNA and protein levels may be one of the main mechanisms for SFN inducing apoptosis of HepG-2 cells.