MG132对肝癌细胞BEL7402凋亡及超微结构影响实验探究.doc

MG132对肝癌细胞BEL7402凋亡及超微结构影响实验探究 　 作者：曲春媚 李伯勤 刘倩 毛海婷 张玲 【摘要】目的：探讨泛素蛋白酶体通路阻断剂MG132对肝癌细胞BEL7402细胞凋亡和超微结构的影响。方法：以5和10μmol / L MG132分别处理人肝癌BEL7402细胞24和48h，用流式细胞术检测细胞凋亡；DNA片段分析进一步证实凋亡存在，Western 印迹检测Bax蛋白表达，比色法测 Caspase3活性变化，用透射电镜观察细胞超微结构的变化。结果：随着MG132浓度的增加和处理时间延长，细胞凋亡率增加；细胞DNA抽提电泳发现特征性凋亡梯状条带；Bax蛋白表达增加；Caspase3活化；电镜观察到典型凋亡细胞，线粒体等细胞器的形态变化与MG132浓度和作用时间有关。结论：蛋白酶体抑制剂MG132可诱导肝癌细胞BEL7402凋亡；线粒体损伤、Bax蛋白上调、Caspase3激活可能在MG132诱导BEL7402细胞凋亡起重要作用。 【关键词】癌，肝细胞#12539;超微结构#12539;MG132#12539;凋亡 Effect of MG132 on ultramicrostructure and apoptosis of human hepatocelluar cancer cell line BEL7402 【ABSTRACT】Objective:To explore the mechanism of the apoptosis of human hepatocellular cancer cells BEL7402 induced by MG132.Method:BEL7402 cells were treated with MG132,apoptosis rate was detected with flow cytometry (FCM),DNA fragment analysis was used for confirming the presence of apoptosis,expression of Bax was dectected by Western blotting,the activity of caspase3 was determined by colorimetric assay,the morphologic change was observed by transmission electron microscope.Result:Cell apoptosis percentage of BEL7402 cell increased with raised concentrations and prolonged treatment of MG132,agarose electrophoresis showed marked ladder,expression of Bax was upregulated,caspase3 was activited,the typic structure of apoptosis cells was found,different features of ultrastructure changes of mitochondria and other organellae were observed in cells exposed to different concentration and treating time of MG132.Conclusion:Proteasome inhibitor MG132 can induce apoptosis of BEL7402 cells,Bax protein and caspase3 and mitochondria injury were responsible for apoptosis of BEL7402 cell induced by MG132. 【KEY WORDS】Carcinoma,hepatocellular#12539;Ultrastructure#12539;MG132#12539;Apoptosis 泛素蛋白酶体途径是细胞内蛋白降解的一个主要途径，这些蛋白包括转录因子，肿瘤抑制蛋白，原癌基因等。抑制泛素蛋白酶体途径，可使细胞生长抑制甚至凋亡［1］。研究发现，蛋白酶体抑制剂可诱导多种肿瘤细胞凋亡［2～4］，本研究探讨蛋白酶体抑制剂MG132（ZLeuLeuLeuCHO）对肝癌细胞BEL7402凋亡及超微结构的影响。 1 材料与方法 1.1 材料 1.1.1 试剂 MG132购自Calbiochem公司，RPMI1640培养基购自GIBCO公司，