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目的:探讨内质网应激在蛋白酶抑制剂MG-132诱导人舌鳞癌细胞株Tca-8113细胞凋亡中的作用。方法:使用含10%胎牛血清的RPMI-1640培养基常规培养Tca-8113细胞,实验分为5组,3个实验组分别加入MG-132,终浓度为10.0、20.0、30.0μmol/L;阳性对照组加入毒胡萝卜素,终浓度为5.0μmol/L;阴性对照组加入1640培养液。培养24h后,Hoechst 33258染色法观察凋亡细胞形态学变化;Annexin V-FITC/PI双染法检测细胞凋亡率,RT-PCR检测GRP78 mRNA,Western印迹检测caspase-12蛋白的表达,ELISA法检测人泛素蛋白连接酶E3浓度。应用SPSS16.0软件包对结果进行统计学分析。结果:MG-132作用Tca-8113细胞24 h后,细胞出现典型的凋亡形态;MG-132实验组细胞凋亡率随MG-132浓度升高而逐渐升高,存在浓度依赖性;MG-132组GRP78的mRNA及caspase-12蛋白表达增强;MG-132组人泛素连接酶E3的浓度分别为28.75±2.28、18.16±0.65、8.85±0.72。结论:MG-132可通过内质网应激途径诱导Tca-8113细胞的凋亡,MG-132可抑制人泛素连接酶E3的表达。
AIM: To investigate the role of endoplasmic reticulum stress in the apoptosis of human tongue squamous carcinoma cell line Tca-8113 induced by protease inhibitor MG-132. Methods: Tca-8113 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. The experiment was divided into 5 groups. MG-132 and MG-132 were respectively added into the three experimental groups. The final concentration was 10.0, 20.0 and 30.0μmol / Positive control group added thapsigargin, the final concentration of 5.0μmol / L; negative control group added 1640 medium. The morphology of apoptotic cells was observed by Hoechst 33258 staining and the apoptosis rate was detected by Annexin V-FITC / PI double staining. The expression of GRP78 mRNA was detected by RT-PCR and the expression of caspase-12 protein was detected by Western blot. Human ubiquitin ligase E3 concentration was measured. SPSS16.0 software package for statistical analysis of the results. Results: MG-132 cells treated with Tca-8113 cells showed typical apoptotic morphology. The apoptosis rate of MG-132 cells increased gradually with the concentration of MG-132 in a concentration-dependent manner. MG- 132 GRP78 mRNA and caspase-12 protein expression increased; MG-132 group ubiquitin ligase E3 concentrations were 28.75 ± 2.28,18.16 ± 0.65,8.85 ± 0.72. Conclusion: MG-132 induces the apoptosis of Tca-8113 cells through endoplasmic reticulum stress, and MG-132 inhibits the expression of human ubiquitin ligase E3.