X-连锁凋亡抑制蛋白在顺铂诱导的喉癌Hep-2细胞凋亡中的作用

目的 探讨X-连锁凋亡抑制蛋白(x-linked inhibitor of apoptpsis protein,XIAP)在顺铂诱导喉癌Hep-2细胞凋亡中的作用和机制.方法 体外培养的Hep-2细胞,选择不同浓度的顺铂以不同时间作用于培养的细胞.利用结晶紫法、RT-PCR、流式细胞分析技术研究不同时间、浓度下细胞凋亡的发生率和XIAP、mRNA的表达及两者的相互关系.结果 正常状态下,Hep-2细胞为贴壁细胞,凋亡率低.顺铂作用后细胞凋亡率和XIAP表达水平发生明显变化,表现为XIAP表达水平的下降伴Hep-2细胞凋亡率的升高.不同药物浓度及不同时间点间相比较,细胞抑制率、XIAP的表达及凋亡率差异均有显著性意义.RT-PCR终产物分析表明,随着加药浓度的增加及药物作用时间的延长,XIAP的mRNA水平逐渐降低.相关性分析显示细胞抑制率、凋亡率与药物浓度及药物的作用时间成正相关,而XIAP的表达与药物浓度及作用时间呈负相关,XIAP的表达与调亡率呈负相关.结论 顺铂引起细胞死亡的主要方式是细胞凋亡.XIAP及mRNA在喉癌Hep2细胞中的表达与顺铂的作用浓度及作用时间有双相依赖性.通过诱发XIAP表达水平的降低使细胞的凋亡率升高是顺铂杀伤肿瘤的作用机制之一.

Role of inhibitor of apoptosis protein XIAP in cisplatin-induced apoptosis of Hep-2 laryngeal carcinoma cells

OBJECTIVE To investigate the role of XIAP in cisplatin-induced apoptosis and its possible mechanism. METHODS Hep-2 cells were treated with different concentrations of cisplatin for different times. Using crystal violet assay, RT-PCR and flow cytometry (FCM), we investingated the rate of the cell apoptosis and the expression of XIAP protein and mRNA at different times after treating with different concentrations of cisplatin. RESULTS Hep-2 cells were adherent and in normal survival condition with very low apoptosis rate. After treating with cisplatin, the rate of inhibition, the expression of XIAP protein and the rate of apoptosis were remarkably different between different concentrations and times. The expression of XIAP protein was down regulated accompanied by the augmentation of the rate of Hep-2 cell apoptosis. The products of RT-PCR were analyzed, demonstrating that the XIAP mRNA was down regulated with the increased concentrations of cisplatin overtime. Correlation analysis showed the rate of inhibition and the rate of apoptosis were positively correlated with increased concentrations of cisplatin for different times, however, the expression of XIAP protein was negatively correlated. The expression of XIAP protein and the rate of apoptosis were conversely correlated. CONCLUSION The most important pathway that cisplatin induces cell death is apoptosis. The levels of expression of XIAP proteinand mRNA in Hep2 cells are time-and concentration-dependent after treating with cisplatin. Down-regulation of the XIAP protein expression to augment the rate of apoptosis is one of the mechanisms for the cisplatin tokill the carcinoma cells.