摘要
目的:体外观察纺锤体驱动蛋白Eg5抑制剂S-三甲基-L-半胱氨酸(STLC)对人膀胱尿路上皮癌BIU-87细胞Eg5基因表达和凋亡的影响。方法:分别采用流式细胞术、四甲基偶氮唑盐微量酶反应(MTT)比色法、TUNEL染色法、逆转录-聚合酶链反应(RT-PCR)和Western Blotting法检测STLC对人膀胱癌BIU-87细胞的细胞周期、生长抑制率、凋亡指数、Eg5 mRNA及其蛋白相对表达水平的影响。结果:将STLC与BIU-87细胞共孵育,不同时间点收集细胞,可见随着STLC作用时间延长至16 h,细胞有丝分裂周期中G2/M期的BIU-87细胞明显增多(29.6%),且细胞发生凋亡的比例也有明显增加的趋势。统计分析显示,与0 h相比,STLC处理16 h时细胞凋亡比例升高,差异有统计学意义(P=0.020)。去除STLC后再培养,于不同时间点收集细胞,结果表明STLC去除后BIU-87细胞周期进程开始恢复,与去除1 h时G2/M期细胞比例(34.84%)相比,去除3 h后G2/M期细胞比例(24.9%)显著降低,差异有统计学意义(P=0.001);细胞凋亡比例差异也有统计学意义(P=0.049)。与对照组相比,STLC处理组细胞增殖抑制率和凋亡指数均显著升高,差异有统计学意义(P<0.001);与对照组相比,STLC处理组Eg5 mRNA和蛋白的表达水平均显著降低,差异有统计学意义(P<0.001)。结论:STLC可有效抑制Eg5基因表达并诱导人膀胱尿路上皮癌BIU-87细胞凋亡,为膀胱尿路上皮癌的药物治疗提供了实验依据和理论基础。
Objective:To observe the effect of S-trimethyl-L-cysteine(STLC),a spindle kinesin Eg5 inhibitor,on Eg5 gene expression and apoptosis in human bladder urothelial carcinoma BIU-87 cells in vitro.Methods:The cell cycle,growth inhibition rate,apoptosis index and Eg5 of human bladder cancer BIU-87 cells were detected by flow cytometry,MTT assay,TUNEL staining,reverse transcription-polymerase chain reaction(RT-PCR)and Western Blotting,respectively Effect of relative expression levels of mRNA and protein.Results:BIU-87 cells were coincubated with STLC,and cells were collected at different time points.As the treatment time of STLC extended to 16 h,the number of BIU-87 cells in G2/M phase of mitotic cycle increased significantly(29.6%),and the proportion of cells undergoing apoptosis also showed a significant increase trend.Statistical analysis showed that compared with 0 h,the proportion of apoptosis was increased at 16 h of STLC treatment,and the difference was statistically significant(P=0.020).Cells were collected at different time points after STLC removal.The results showed that the cycle progression of BIU-87 cells began to recover after STLC removal.Compared with the proportion of cells in G2/M phase(34.84%)at 1 h removal,the proportion of cells in G2/M phase(24.9%)at 3 h removal was significantly reduced.The difference was statistically significant(P=0.001).The proportion of apoptosis was also significantly different(P=0.049).Compared with the control group,the proliferation inhibition rate and apoptosis index of STLC treatment group were significantly increased(P<0.001).Compared with the control group,the expression levels of Eg5 mRNA and protein in the STLC treatment group were significantly decreased(P<0.001).Conclusion:STLC can effectively inhibit the expression of Eg5 gene and induce apoptos is of human bladder urothelial carcinoma BIU-87 cells,which provides experimental and theoretical basis for drug treatment of bladder urothelial carcinoma.
作者
曹正国
田超
钟苏权
周琳雄
陈桂柳
CAO Zhengguo;TIAN Chao;ZHONG Suquan;ZHOU Linxiong;CHEN Guiliu(Second Department of Urology,North Guangdong People’s Hospital,Shantou University Medical College,Shaoguan Guangdong 512026)
基金
广东省自然科学基金资助项目(编号S2011010004230)
韶关市科技计划资助(编号2019sn004)
韶关市卫健局资助项目(编号Y20144)。
作者简介
曹正国(1975-),男,汉族,博士,硕士生导师,主任医师,研究方向:泌尿系肿瘤的临床与基础研究,E-mail:19655975@qq.com。
