氯化镉对ZR75-1细胞DNA错配修复活性的抑制效应

The inhibitory effect of CdCl_2 on DNA mismatch repair activity in ZR75-1 cells

目的:研究氯化镉(Cd Cl2)暴露对人乳腺癌细胞ZR75-1 DNA错配修复(DNA MMR)活性的影响。方法:用0、10、20、30、40、50 μmol/L的Cd Cl2对ZR75-1细胞染毒48 h和72 h后,用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测各组ZR75-1细胞的存活率。以5、10、15 μmol/L的Cd Cl2分别作用于ZR75-1细胞48 h后,各组平行转染同源、异源质粒,通过流式细胞术检测相对荧光表达率来评估各组ZR75-1细胞的DNA MMR活性;同时利用实时荧光定量PCR技术检测Cd Cl2暴露后DNA MMR活性相关基因MLH1、MSH2、MSH6、PMS1 m RNA的表达。结果:MTT结果显示,与对照组相比,10、20 μmol/L Cd Cl2对ZR75-1细胞的存活率无显著影响(P>0.05),而30、40、50 μmol/L Cd Cl2对细胞有显著毒性作用(P<0.05),且表现出浓度和时间依赖效应。流式细胞术检测显示,与对照组相比,5、10、15 μmol/L Cd Cl2暴露均显著降低了ZR75-1细胞的DNA MMR活性(P<0.05)。荧光定量PCR技术检测结果显示,5、10、15 μmol/L Cd Cl2处理组中MLH1、MSH2、MSH6、PMS1 m RNA表达水平总体上升,变化趋势均为随Cd Cl2暴露浓度增加先上升后下降。结论:对细胞存活率无影响的Cd Cl2暴露可显著抑制ZR75-1细胞的DNA MMR活性,并上调DNA MMR活性相关基因MLH1、MSH2、MSH6、PMS1 m RNA的表达水平。

Objective： To study the effects of CdCl2 on the DNA mismatch repair （DNA MMR） activity in human breast cancer cells ZR75-1. Methods： MTT assay was used to detect the survival rate of ZR75-1 cells exposed 48 and 72 h to 0, 10, 20, 30, 40, 50 μmol/L CdCl2. The cells from 5, 10, 15 μmol/L CdC12 exposure （48 h） and control group were parallelly transfected with homoduplex and heteroduplex plasmids, flow cytometry analysis was then carried out to quantitatively measure the relative EGFP expression （indirectly reflected DNA MMR activity） in ZR75-1 cells of each groups. Meanwhile, real-time fluorescent quantitative PCR technique was used to detect the mRNA expression of DNA MMR related genes （MLH 1, MSH2, MSH6 and PMS 1） after expo- sure 48 h to 5, 10, 15 μmol/L CdC12. Results： 10, 20 p.mol/L CdCI2 exhibited no influence on ZR75-1 cell surviv- al （P〉0.05）, while cells exposed to 30, 40 and 50 μmol/L CdC12 displayed significant mortality which showed ob- vious dose-dependent and time-dependent manner （P〈0.05）. Compared with control group, 5, 10 and 15 pmol/L CdCI2 exposure induced significant inhibition of DNA MMR activity in ZR75-1 cells （P〈0.05）. Real-time PCR results showed that after exposure to 5, 10 and 15 μmol/L CdCl2 for 48 h, the mRNA expression of detected genes （MLH1, MSH2, MSH6 and PMS1） was increased as a whole, which presented down after rising first with CdCl2 exposure concentration increase. Conclusion： Low dose of CdCl2 exposure which showed no effect on cell survival can represse DNA MMR activity in ZR75-1 cells, and up-regnlate the mRNA expression of DNA MMR related genes.