中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2015年
3期
206-211
,共6页
洪文旭%黄爱博%许华%张航%王宏菊%赵琼晖%叶金波%刘建军
洪文旭%黃愛博%許華%張航%王宏菊%趙瓊暉%葉金波%劉建軍
홍문욱%황애박%허화%장항%왕굉국%조경휘%협금파%류건군
三氯乙烯%DNA甲基化%人正常肝细胞%SET缺陷%DNA甲基转移酶
三氯乙烯%DNA甲基化%人正常肝細胞%SET缺陷%DNA甲基轉移酶
삼록을희%DNA갑기화%인정상간세포%SET결함%DNA갑기전이매
Trichloroethylene%DNA methylation%Human hepatic L-02 cells%SET deficiency%DNA methyltransferases
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.
目的 比較三氯乙烯(trichloroethylene)誘導人正常肝細胞(L-02細胞)和SET基因缺陷L-02細胞(SET缺陷細胞)中DNA甲基化相關指標的變化,探討SET與三氯乙烯誘導的錶觀遺傳調控作用之間的關繫.方法 選用前期建立的SET缺陷細胞為研究對象,使用三氯乙烯分彆對L-02細胞和SET缺陷細胞進行處理,檢測細胞增殖水平、細胞DNA甲基化水平和DNA甲基轉移酶(DNAmethyltransferases,DNMTs)活性的變化,通過Western blot法從蛋白水平分析DNMTs(DNMT1、DNMT3a、DNMT3b)的錶達變化.結果 三氯乙烯處理L-02細胞和SET缺陷細胞24 h後,兩種細胞的增殖水平均呈現下降趨勢.使用0、1.0、2.0、4.0和8.0 mmol/L的三氯乙烯處理細胞後,L-02細胞的相對增殖水平分彆為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缺陷細胞的相對增殖水平分彆為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的三氯乙烯處理L-02細胞和SET缺陷細胞24 h後,L-02細胞的DNA甲基化水平分彆為3.77 ±0.08、3.48 ±0.08、3.38 ±0.10、3.14 ±0.15、2.91 ±0.07,呈下降趨勢(F =212.87,P＜0.001);SET缺陷細胞DNA甲基化水平分彆為3.77 ±0.15、3.57 ±0.15、3.30±0.11、3.35±0.13呈下降趨勢(F =79.32,P＜0.001).L-02細胞經0、1.0、2、0、4.0、8.0 mmol/L的三氯乙烯處理24 h後,DNMT1蛋白的相對錶達量分彆為1.00±0.03、1.28±0.04、1.20±0.04、1.62±0.05、1.43 ±0.04,錶達升高(F=103.00,P＜0.001);而在SET缺陷細胞中DNMT1的相對錶達量分彆為1.00±0.04、0.96±0.02、1.19 ±0.05、0.85±0.03、0.83±0.03,齣現下降(F =44.18,P＜0.001).結論 SET缺陷可以明顯抑製三氯乙烯暴露引起的L-02細胞增殖水平及DNMTs活性的下降,改變DNMT1錶達水平的變化趨勢,提示SET參與瞭在三氯乙烯誘導下的肝毒性作用和錶觀遺傳學調控機製.
목적 비교삼록을희(trichloroethylene)유도인정상간세포(L-02세포)화SET기인결함L-02세포(SET결함세포)중DNA갑기화상관지표적변화,탐토SET여삼록을희유도적표관유전조공작용지간적관계.방법 선용전기건립적SET결함세포위연구대상,사용삼록을희분별대L-02세포화SET결함세포진행처리,검측세포증식수평、세포DNA갑기화수평화DNA갑기전이매(DNAmethyltransferases,DNMTs)활성적변화,통과Western blot법종단백수평분석DNMTs(DNMT1、DNMT3a、DNMT3b)적표체변화.결과 삼록을희처리L-02세포화SET결함세포24 h후,량충세포적증식수평균정현하강추세.사용0、1.0、2.0、4.0화8.0 mmol/L적삼록을희처리세포후,L-02세포적상대증식수평분별위100.00 ±2.70、83.34±2.38、75.56±4.51、71.67±2.77、66.67±1.63(F=58.29,P＜0.001);SET결함세포적상대증식수평분별위101.12±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、4.0화8.0 mmol/L적삼록을희처리L-02세포화SET결함세포24 h후,L-02세포적DNA갑기화수평분별위3.77 ±0.08、3.48 ±0.08、3.38 ±0.10、3.14 ±0.15、2.91 ±0.07,정하강추세(F =212.87,P＜0.001);SET결함세포DNA갑기화수평분별위3.77 ±0.15、3.57 ±0.15、3.30±0.11、3.35±0.13정하강추세(F =79.32,P＜0.001).L-02세포경0、1.0、2、0、4.0、8.0 mmol/L적삼록을희처리24 h후,DNMT1단백적상대표체량분별위1.00±0.03、1.28±0.04、1.20±0.04、1.62±0.05、1.43 ±0.04,표체승고(F=103.00,P＜0.001);이재SET결함세포중DNMT1적상대표체량분별위1.00±0.04、0.96±0.02、1.19 ±0.05、0.85±0.03、0.83±0.03,출현하강(F =44.18,P＜0.001).결론 SET결함가이명현억제삼록을희폭로인기적L-02세포증식수평급DNMTs활성적하강,개변DNMT1표체수평적변화추세,제시SET삼여료재삼록을희유도하적간독성작용화표관유전학조공궤제.
Objective 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.
