微囊藻毒素-LR对小鼠肝细胞DNA甲基化的影响

Effects of microcystin-LR on DNA methylation in mouse hepatocytes

目的研究微囊藻毒素-LR(MC-LR)暴露对小鼠肝细胞DNA总体甲基化水平及DNA甲基转移酶(DNMTs)mRNA表达的影响。方法将40只健康SPF级昆明小鼠随机分为4组,分别为对照组(含0.02%DMSO)和5、10、20μg/kg MCLR染毒组,每组10只,雌雄各半。采用腹腔注射方式进行染毒,连续染毒20 d。采用高效液相色谱(HPLC)法检测小鼠肝细胞DNA中的脱氧胞苷和5-甲基脱氧胞苷含量,并计算DNA的总体甲基化水平;采用实时荧光定量PCR法检测DNA甲基转移酶DNMT1、DNMT3A和DNMT3B m RNA的表达水平。结果 10、20μg/kg MC-LR染毒组小鼠肝细胞DNA总体甲基化水平,DNMT1、DNMT3A及DNMT3B mRNA的表达水平均低于对照组,差异有统计学意义(P<0.05)。小鼠肝细胞DNA总体甲基化水平与DNMT1、DNMT3A、DNMT3B mRNA表达水平均呈正相关(P<0.05)。结论 MC-LR可抑制小鼠肝细胞DNMT1、DNMT3A和DNMT3B mRNA的表达,降低DNA总体甲基化水平。

Objective To investigate the effects of microcystin-LR（MC-LR） on the global DNA methylation level and m RNA expression of DNA methyltransferase in mouse hepatocytes under the conditions of short-term and continuous exposure.Methods Forty Kunming mice were randomly divided into four groups： in the control group,0.005 ml/g of saline containing 0.02% dimethyl sulfoxide was intraperitoneal injected,whereas in the three exposure groups MC-LR were administered at doses of 5,10 and 20 μg/kg body weight for 20 days. DNA was extracted from mouse hepatocytes,global DNA methylation level was determined by high performance liquid chromatography（HPLC）. RNA was extracted and reverse transcribed into cDNA,expression of DNMT1,DNMT3A and DNMT3B mRNA were measured by real time PCR. Results Global DNA methylation levels in the 10 and 20 μg/kg body weight MC-LR groups were significantly lower than that of the control group（P〈0.05）. The expressions of DNMT1,DNMT3A and DNMT3B m RNA in the 10 and 20 μg/kg body weight MC-LR group were significantly lower compared with the control group（P〈0.05）. The global DNA methylation levels were positively correlated with expression of DNMT1,DNMT3A and DNMT3B m RNA. Conclusion MC-LR may induce the down-regulation of DNMT1,DNMT3 A,DNMT3B m RNA as well as a decrease in global DNA methylation levels in mice hepatocytes.