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目的:研究衣霉素(tunicamycin,TM)上调胃癌细胞对肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)诱导凋亡敏感性的作用机制。方法:采用碘化丙啶染色的FCM法检测TM(1μmol/L)与TRAIL(100μg/L)单独或联合作用3、6、16、24和36h时对SGC-7901细胞凋亡率的影响;FCM法检测TM处理前后细胞表面TRAIL受体1(TRAIL receptor1,TRAIL-R1)、-R2、-R3和-R4的表达情况;实时荧光定量-PCR法检测TRAIL-R2mRNA的表达情况;蛋白质印迹法检测葡萄糖调节蛋白78(glucose-regulated protein 78kDa,GRP78)和CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)的表达水平;RT-PCR检测X盒结合蛋白(X-box binding protein1,XBP1)mRNA的剪接情况。结果:TM单独作用引起的SGC-7901细胞的凋亡率低,TM和TRAIL联合作用能有效提高SGC-7901细胞的凋亡率;TM能明显上调SGC-7901细胞表面TRAIL-R2的表达水平,而对TRAIL-R1、-R3和-R4却无明显影响;TRAIL-R2mRNA的表达水平随TM作用时间延长而相应升高;GRP78蛋白的上调和XBP-1mRNA的剪接活化证实了TM可诱导未折叠蛋白反应(unfolded protein response,UPR)的发生;CHOP蛋白的表达水平也在TM作用后上调。结论:TM通过诱导UPR上调TRAIL-R2的表达,从而增加胃癌细胞对TRAIL的敏感性,CHOP介导了TRAIL-R2的上调作用。
Objective: To investigate the mechanism of tunicamycin (TM) upregulating the sensitivity of gastric cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Methods: The apoptosis rate of SGC-7901 cells treated with TM (1μmol / L) and TRAIL (100μg / L) alone or in combination for 3, 6, 16, 24 and 36 hours was detected by propidium iodide staining. The expression of TRAIL receptor1 (TRAIL-R1), -R2, -R3 and -R4 on the cell surface was detected by FCM. The expression of TRAIL-R2 mRNA was detected by real-time fluorescence quantitative PCR and Western blotting The expression levels of glucose-regulated protein 78kDa (GRP78) and CCAAT / enhancer-binding protein homologous protein (CHOP) were detected by RT-PCR. -box binding protein1, XBP1) mRNA splicing. Results: The apoptosis rate of SGC-7901 cells induced by TM alone was low, the combination of TM and TRAIL could effectively increase the apoptosis rate of SGC-7901 cells; TM significantly increased the expression of TRAIL-R2 on SGC-7901 cells, But not TRAIL-R1, -R3 and -R4. The expression of TRAIL-R2 mRNA increased with the prolongation of TM effect. The up-regulation of GRP78 protein and splicing activation of XBP-1 mRNA confirmed that TM induced unfolded (Unfolded protein response, UPR) occurred; CHOP protein expression levels are also up after the TM effect. Conclusion: TM induces UPR upregulation of TRAIL-R2, thereby increasing the sensitivity of gastric cancer cells to TRAIL. CHOP mediates the up-regulation of TRAIL-R2.