DNA甲基化对邻苯二甲酸单乙基己酯致小鼠睾丸间质细胞损伤的作用

The Role of DNA Methylation in the Reproductive Damage of Mouse Testicular Stromal Cells Induced by Mono-(2-ethylhexyl)Phthalate

目的探讨邻苯二甲酸单乙基己酯[mono-(2-ethylhexyl)phthalate,MEHP]染毒对小鼠睾丸间质细胞(TM-3)DNA甲基化和凋亡水平的影响。方法体外培养小鼠睾丸间质细胞至对数生长期,预实验毒物浓度梯度设为0、50、100、200、400、800μmol·L^(-1),24 h染毒后,采用CCK-8法检测细胞活性,采用Graphpad prism 6.0计算细胞半数抑制剂量(IC50),并最终确定染毒剂量为0(对照组)、200、400、800μmol·L^(-1);光镜观察细胞形态学变化,采用5-甲基胞嘧啶抗体(5-methylcytosine,5-mc)免疫荧光法检测细胞总甲基化水平;采用RT-PCR检测甲基转移酶(DNMTs)中DNMT1、DNMT3A、DNMT3B mRNA表达水平;采用Hoechst荧光染色检测细胞凋亡水平。结果CCK-8结果显示:随着染毒剂量的增加,细胞存活率逐渐下降(P<0.05);光镜观察显示:正常细胞呈梭形、多边形等不规则形状生长,随着染毒剂量的增加,贴壁细胞数量逐渐减少,细胞核呈现空泡化,条索状细胞增多,且漂浮的死亡细胞数逐渐增加;5-mc免疫荧光结果显示:与对照组相比,200μmol·L^(-1)组荧光信号最强,400μmol·L^(-1)组次之,800μmol·L^(-1)组最弱(P均<0.01),不同浓度MEHP暴露,均可引起TM-3细胞总甲基化水平降低(P均<0.01);RT-PCR结果显示:与对照组相比,200μmol·L^(-1)组DNMT1 mRNA(1.15±0.08)、DNMT3B mRNA(2.28±0.15)相对表达水平高于对照组(P<0.05或<0.01);400μmol·L^(-1)组DNMT1 mRNA(0.83±0.05)低于对照组(P<0.01),DNMT3A mRNA(1.91±0.44)和DNMT3B mRNA(2.50±0.09)表达水平高于对照组(P<0.05或<0.01);800μmol·L^(-1)组DNMT1 mRNA(0.50±0.08)低于对照组,DNMT3A mRNA(2.40±0.51)高于对照组(P<0.01);Hoechst33258荧光染色显示:与对照组相比,不同浓度MEHP暴露,均可诱导TM-3细胞凋亡(P均<0.01);400、800μmol·L^(-1)两组可以观察到明显的染色质固缩,核浓染现象,凋亡细胞数目逐渐增多。结论MEHP可引起TM-3细胞发生异常的甲基化修饰,进而导致细胞凋亡。

Objective To investigate the effects of mono-(2-ethylhexyl)phthalate(MEHP)on DNA methylation and apoptosis of mouse leydig cells(TM-3)in mice.Methods In vitro culture of mouse leydig cells to logarithmic growth phase,the concentration gradient of pre experiment poison was set at 0(control group),50,100,200,400,800μmol·L^(-1).After 24 hours of exposure,CCK-8 method was used to detect the cell activity,and Graphpad prism 6.0 was used to calculate the half inhibitory dose(IC50),and finally determined the dose of 0(control group),200,400 and 800μmol·L^(-1).The total methylation level was detected by 5-methylcytosine(5-mc)immunofluorescence assay.The mRNA expression levels of DNMT1,DNM3A and DNM3B were detected by RT-PCR.The apoptosis level was detected by Hoechst fluorescent staining.Results The results of CCK-8 showed that the survival rate of cells decreased gradually(P<0.05)with the increase of the exposure dose.Light microscope observation showed that normal cells grew in irregular shapes such as fusiform and polygonal shapes.As the exposure dose increased,the number of adherent cells gradually decreased,the nucleus showed vacuolation,the number of strand-like cells increased,and the number of floating dead cells gradually increased.5-mc immunofluorescence results showed that compared with the control group,the fluorescence signal of the 200μmol·L^(-1) group was the strongest,followed by the 400μmol·L^(-1) group,and the 800μmol·L^(-1) group the weakest.The difference was statistically significant(P<0.01).Total methylation levels of TM-3 cells decreased with MEHP exposure(P<0.01).The results of RT-PCR showed that compared with the mRNA level of the control group,the relative expression levels of DNMT1 mRNA(1.15±0.08)and DNMT3B mRNA(2.28±0.15)in the 200μmol·L^(-1) group were higher than those in the control group(P<0.05 or<0.01),the expression level of DNMT3A mRNA(1.03±0.12)was not statistically different between the two groups(P>0.05);the 400μmol·L^(-1) group DNMT1 mRNA(0.83±0.05)was lower than the control group(P<0.01).The expression levels of DNMT3A mRNA(1.91±0.44)and DNMT3B mRNA(2.50±0.09)were higher than those of the control groups(P<0.05 or<0.01).The 800μmol·L^(-1) group DNMT1 mRNA(0.50±0.08)was lower than the control group.The expression levels of DNMT3A mRNA(2.40±0.51)were higher than those of the control groups(P<0.01).Hoechst33258 fluorescence staining showed that,different cancentrations of MEHP exposure induced TM-3 cell apoptosis(P all<0.01),400,800μmol·L^(-1) two groups,can observe obvious chromatin fixation,nuclear thickening phenomenon,the number of apoptotic cells gradually increased.Conclusion MEHP can cause abnormal methylation of TM-3 cells,and lead to apoptosis.