摘要目的 比较三氯乙烯(trichloroethylene)诱导人正常肝细胞(L-02细胞)和SET基因缺陷L-02细胞(SET缺陷细胞)中DNA甲基化相关指标的变化,探讨SET与三氯乙烯诱导的表观遗传调控作用之间的关系.方法 选用前期建立的SET缺陷细胞为研究对象,使用三氯乙烯分别对L-02细胞和SET缺陷细胞进行处理,检测细胞增殖水平、细胞DNA甲基化水平和DNA甲基转移酶(DNAmethyltransferases,DNMTs)活性的变化,通过Western blot法从蛋白水平分析DNMTs(DNMT1、DNMT3a、DNMT3b)的表达变化.结果 三氯乙烯处理L-02细胞和SET缺陷细胞24 h后,两种细胞的增殖水平均呈现下降趋势.使用0、1.0、2.0、4.0和8.0 mmol/L的三氯乙烯处理细胞后,L-02细胞的相对增殖水平分别为100.00 ±2.70、83.34±2.38、75.56±4.51、71.67±2.77、66.67±1.63(F=58.29,P＜0.001);SET缺陷细胞的相对增殖水平分别为101.12±1.67、85.01±2.33、79.44±1.67、78.337±3.89、76.11 ±3.33(F=42.41,P＜0.001).0、1.0、2.0、4.0和8.0 mmol/L的三氯乙烯处理L-02细胞和SET缺陷细胞24 h后,L-02细胞的DNA甲基化水平分别为3.77 ±0.08、3.48 ±0.08、3.38 ±0.10、3.14 ±0.15、2.91 ±0.07,呈下降趋势(F =212.87,P＜0.001);SET缺陷细胞DNA甲基化水平分别为3.77 ±0.15、3.57 ±0.15、3.30±0.11、3.35±0.13呈下降趋势(F =79.32,P＜0.001).L-02细胞经0、1.0、2、0、4.0、8.0 mmol/L的三氯乙烯处理24 h后,DNMT1蛋白的相对表达量分别为1.00±0.03、1.28±0.04、1.20±0.04、1.62±0.05、1.43 ±0.04,表达升高(F=103.00,P＜0.001);而在SET缺陷细胞中DNMT1的相对表达量分别为1.00±0.04、0.96±0.02、1.19 ±0.05、0.85±0.03、0.83±0.03,出现下降(F =44.18,P＜0.001).结论 SET缺陷可以明显抑制三氯乙烯暴露引起的L-02细胞增殖水平及DNMTs活性的下降,改变DNMT1表达水平的变化趋势,提示SET参与了在三氯乙烯诱导下的肝毒性作用和表观遗传学调控机制.

abstractsObjective To compare the DNA methylation-related alteration induced by trichloroethylene (TCE) in human hepatic L-02 cells (L-02 cells) and SET deficient cells,and reveal the role of SET on the mechanisms in TCE-induced epigenetic pathway.Methods The L-02 cells and preestablished SET deficient cells were treated with different TCE concentrations,and the changes of total cell viability,DNA methylation level and DNA methyltransferases (DNMTs) activity were measured,respectively.In addition,the TCE-induced alteration in the protein expression of DNMT1,DNMT3a and DNMT3b were analyzed by Western blotting.Results After treatment with TCE for 24 h,the cell proliferation level was significantly decreased in both cell lines.When concentrations of TCE were 0,1.0,2.0,4.0 and 8.0 mmol/L,the proliferation levels of L-02 cells were 100.00 ± 2.70,83.34 ± 2.38,75.56±4.51,71.67 ± 2.77 and 66.67 ± 1.63,respectively (F =58.29,P ＜ 0.001);the cell proliferation levels of SET deficient cells were 101.12 ± 1.67,85.01 ±2.33,79.44 ± 1.67,78.337 ±3.89 and 76.11 ± 3.33,respectively (F =42.41,P ＜ 0.001).When concentration of TCE reached 4.0 mmol/L,the difference of cell proliferation level between two groups was statistically significant (t =-3.51;P =0.013).After treated by TCE for 24 h,the global DNA methylation significantly decreased in both cell lines (F value was 212.87 and 79.32,respectively,P ＜ 0.001).The difference between two groups was not statistically significant.After treated by TCE for 24 h,the methyltransferases activities were significantly decreased in both cell cells (F values were 77.92 and 113.80,respectively,P ＜ 0.001).The SET deficiency could inhibit the decrease of methyltransferases activity under TCE treatment.When the concentration of TCE reached 8.0 mmol/L,the enzymatic activity of L-02 cells and SET deficient cells decreased to 67.61％ ± 2.85％ and 72.97％ ± 1.94％,respectively.The difference between two groups was statistically significant (t =-3.94,P =0.008).After treated with TCE for 24 h,concentrations of TCE were 0,1.0,2.0,4.0 and 8.0 mmol/L,and the relative protein levels of DNMT1 in normal L-02 cells increased significantly to 1.00 ± 0.03,1.28 ± 0.04,1.20 ± 0.04,1.62 ± 0.05,1.43 ± 0.04 (F =103.00,P ＜0.001);In SET deficient cells,the expressions of DNMT1 were 1.00 ± 0.04,0.96 ± 0.02,1.19 ± 0.05,0.85 ±0.03,0.83 ±0.03,which was significantly down-regulated under TCE treatment (F =44.18,P ＜ 0.001).Conclusion SET deficiency can significantly attenuate the TCE-induced decreases of cell viability and DNMTs activity,as well as alteration of protein expression of DNMT1 in L-02 cells,which indicated that SET was involved in the mechanism of TCE-induced cytotoxicity and epigenetic pathway in L-02cells.