Cyclin D1 siRNA转染人肝癌细胞的增殖、凋亡变化及其机制

摘要

目的 观察Cyclin D1 siRNA转染的人肝癌细胞增殖、凋亡变化,并探讨其机制.方法 培养人肝癌细胞株Hep3B,将细胞分为空白对照组、阴性对照组和实验组.阴性对照组和实验组分别通过脂质体转染NC siRNA和Cyclin D1 siRNA,转染48 h后收获细胞.采用MTT法检测细胞增殖能力;流式细胞仪观察凋亡细胞并计算细胞凋亡率;免疫组化法检测各组细胞中的Caspase-3蛋白;采用RT-PCR法检测各组细胞中的乙型肝炎病毒x蛋白(HBx)、着色性干皮病基因蛋白D(XPD)、Bcl-2、Bax mRNA;Western blotting法检测各组细胞中的HBx、XPD、Bcl-2、Bax蛋白.结果 与空白对照组和阴性对照组相比,实验组细胞增殖能力减弱、凋亡率升高、Caspase-3相对表达量增高(P均<0.01).与空白对照组和阴性对照组相比,实验组HBx、Bcl-2 mRNA及蛋白表达下调,XPD、Bax mRNA及蛋白表达上调(P均<0.01).结论 Cyclin D1 siRNA转染的人肝癌细胞增殖受到抑制、凋亡增多,其机制可能与HBx、Bcl-2表达下调及XPD、Bax表达上调有关.%Objective To observe the changes of proliferation and apoptosis of human hepatocellular cells transfected with Cyclin D1 siRNA,and to explore the mechanism. Methods The cultured human hepatoma cell line Hep3B was di-vided into the blank control group,negative control group,and experimental group. The negative control group and the ex-perimental group were transfected with NC siRNA and Cyclin D1 siRNA by liposomes,and the cells were harvested after 48 h. MTT was used to detect cell proliferation;the flow cytometry was used to observe apoptotic cells and we calculated apop-tosis rate;the Caspase-3 protein in cells of each group was detected by immunohistochemistry;the mRNA and protein ex-pression levels of hepatitis B virus x protein (HBx),xeroderma pigmentosum D (XPD),Bcl-2,and Bax in cells of each group were detected by RT-PCR and Western blotting,respectively. Results Compared with the blank control group and the negative control group,the cell proliferation ability of the experimental group decreased,the apoptosis rate increased, and the relative expression of Caspase-3 increased (all P < 0. 01). Compared with the blank control group and the negative control group,the mRNA and protein expression levels of HBx and Bcl-2 in the experimental group were down-regulated, while those of XPD and Bax were up-regulated (all P < 0. 01). Conclusion The proliferation of human hepatoma cells transfected with Cyclin D1 siRNA is inhibited and the apoptosis increases,and the mechanism may be related to the down-regulation of HBx and Bcl-2 expression,and up-regulation of XPD and Bax expression.