奥沙利铂诱导人骨髓瘤细胞RPMI8226凋亡机制的实验研究

摘要

本研究探讨奥沙利铂对人骨髓瘤细胞RPMI8226的增殖抑制作用及其分子机制.将奥沙利铂作用于骨髓瘤细胞,用MTT法检测细胞增殖抑制率,光学显微镜和电子显微镜观察细胞形态及超微结构变化,流式细胞术检测细胞凋亡率和细胞周期分布,半定量RT-PCR检测Bcl-2、caspase-8及caspase-3 mRNA表达量的变化,Western blot 检测Bcl-2蛋白表达量变化.结果表明,奥沙利铂可抑制RPMI8226细胞增殖,抑制率呈时间(r=0.979)和浓度(r=0.949)依赖性增强；24 h后在光学显微镜下可见奥沙利铂组细胞数量减少,排列紊乱,细胞形态变得不规则,体积变小,细胞碎片增多,可见凋亡细胞；48 h电子显微镜下可见细胞典型凋亡改变,凋亡小体形成.流式细胞术检测结果显示,奥沙利铂组RPMI8226细胞凋亡率明显增高(P＜0.05)；奥沙利铂作用24 h后,骨髓瘤细胞被阻滞于S期(P＜0.05)；而作用48 h后G0/G1期细胞所占比例明显增高(P＜0.05)；奥沙利铂作用48 h,细胞Bcl-2 mRNA与Bcl-2蛋白表达量未见明显变化,caspase-8及caspase-3 mRNA表达量增加(P＜0.05).结论:奥沙利铂可诱导RPMI8226细胞凋亡,其机制可能与其将细胞阻滞于S期,上调caspase-8、caspase-3 mRNA表达有关；奥沙利铂诱导RPMI8226细胞凋亡可能不通过调节Bcl-2基因表达实现.%This study was aimed to investigate the effects of oxaliplatin on human multiple myeloma cell line RPMI-8226 and its mechanism. The proliferation inhibitory rate of RPMI8226 cells was assayed by MTT, the morphological changes of RPMI-8226 cells were observed by inverted flurescent microscopy and transmission electron microscopy, the apoptosis rate and the cell cycle distribution of RPMI-8226 cells were detected by flow cytometry, The effects of oxaliplatin on the expression of Bcl-2, caspase-8, caspase-3 mRNA were tested by RT-PCR, Bcl-2 protein expression of RPMI-8226 cells was analyzed by Western blot. The results showed that oxaliplatin could inhibit the proliferation of RPMI-8226 cells in time and dose-dependent manners. Cell number in oxaliplatin group was significantly less than that in control group under light microscope, and the growth arrangement was irregular, apoptotic cells could be seen. Under electron microscope, typical apoptotic morphological and ultrastructural changes could be observed. Flow cytometry results showed that oxaliplatin could induce apoptosis of RPMI-8226 cells, the difference have statistical significance(Prn<0.05). Oxaliplatin mainly arrested RPMI-8226 cells in the S phase(P < 0. 05). The expression of Bcl-2 mRNA did not have apparent change, while the expression of caspase-8, caspase-3 mRNA increased (P < 0.05). Western blot results suggested that the expression of Bcl-2 protein had no obvious change. It is concluded that the oxaliplatin can induce apoptosis of RPMI-8226 cells, activating the death receptor pathway and arresting cell cycle may be two of the relatated mechanisms, Bcl-2 gene has unobservable effects in the process of oxaliplatin-induced cell apoptosis.