摘要
目的探讨RAD51抑制剂RI-1对MSH2缺陷结直肠癌细胞系的杀伤作用及可能的作用机制。方法 Western blot法筛选MSH2差异表达的结直肠癌细胞系,MTT法检测不同结直肠癌细胞系对RI-1(10、20、30、40或50μmol/L)的敏感性;构建针对MSH2基因的重组shRNA慢病毒表达载体并感染HT29细胞。RI-1(40μmol/L)处理细胞,流式细胞术检测细胞凋亡的变化;单细胞凝胶电泳实验分析细胞内DNA损伤情况;细胞免疫荧光法检测γ-H2AX foci的形成。结果与对照组相比MSH2缺陷的HCT8细胞有明显细胞凋亡现象(P<0.01);HCT8细胞和HT29 Shmsh2细胞尾部DNA含量、尾长和尾距均较对照组明显增加(P<0.05);HCT8细胞和HT29 Shmsh2细胞内γ-H2AX foci的形成数量较对照组明显增加(P<0.01)。结论 RAD51抑制剂RI-1可能通过增加细胞内DNA损伤参与RI-1对MSH2缺陷肿瘤的杀伤作用。
Objective To explore the lethal action and possible mechanism of RI-1,a RAD51 inhibitor,on MSH2 deficient colorectal cancer cells. Methods The expression of MSH2 protein level was assessed by Western blot,and the sensibility of human colorectal cancer cells to RI-1（ 10,20,30,40 and 50 μmol/L） was measured by MTT method. Lentivirus vectors MSH2-shRNA and Neg-shRNA（ negative control） were constructed and transfected into HT29 cell. Apoptosis and DNA damage of cells treated with RI-1（ 40 μmol/L） were detected by flow cytometry and Single cell gel electrophoresis respectively. In addition,the formation of γ-H2AX foci was analyzed by immunofluorescence. Results Compared with control,MSH2-deficient HCT8 cells had obviously apoptosis（ P〈0. 01）; in HCT8 and HT29 Shmsh2 cells,tail DNA%,tail length,tail moment and olive tail moment were markedly increased（ P〈0. 05）,and the number of γ-H2AX focus were increased（ P〈0. 01）. Conclusions RAD51 inhibitor RI-1 selectively kills MSH2 deficient colorectal cancer cells by increasing DNA damage.
出处
《基础医学与临床》
CSCD
2017年第4期473-478,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(81302803)
天津市应用基础与前沿技术研究计划(14JCYBJC28800)
