污染水产去甲基化表观遗传毒性EGFP评价方法

A novel evaluation method for epigenetic demethylation toxicity of contaminated aquatics based on EGFP reporter

pEGFP-C3质粒经过体外人工甲基化处理后,被转染进入HepG2细胞以构建重组细胞株.以5-AZA为阳性去甲基化毒物与重组细胞共培养,通过亚硫酸氢钠测序法定量检测EGFP基因启动子区甲基化状态,通过实时定量PCR检测EGFP基因表达,借助流式细胞术和荧光摄片定量检测共培养细胞的绿色荧光强度,在DNA甲基化、EGFP基因mRNA表达、GFP蛋白等多个层次研究5-AZA染毒处理与其去甲基化能力和荧光表达改变的响应关系.对天津污染水产的去甲基化能力进行了实际样品测试.结果表明,5-AZA与重组细胞的DNA甲基化、基因表达、蛋白产物变化之间存在显著关联,具有较低的检出浓度和良好的重复性.天津污染海域的水产去甲基化能力较强.本文初步建立了一种污染物去甲基化表观遗传毒性评价方法.

To study a novel evaluation method for demethylation epigenetic toxicity of pollutants based on an artificial recombinant pEGFP-C3 plasmid, pEGFP-C3 plasmid was methylated with M.Sss I in vitro first and then transfected into HepG2 cells. Taking 5-AZA as positive demethylation agent, the levels ofmethylation of the EGFP CMV promoter region, EGFP gene expression and green fluorescence intensity of the recombinant cell lines was quantified with sodium bisulfite sequencing assay, quantitative real-time quantitative PCR and flow cytometry at the time of 24 h after the cells co-cultured with 5-AZA gradients, respectively. A dose-respond relationship was explored between the cells＇ response intensity at the former three levels and the co-cultured 5-AZA. The demethylation ability of the aquatic from polluted area of Tianjin basin was tested with this novel method. Good dose-respond relationships were found between DNA methylation of CMV promoter, EGFP gene expression, green fluorescence intensity of the recombinant cells and 5-AZA. The equation for green fluorescence intensity of the test cells and 5-AZA is y = 0.640lnx ＋ 10.284 with R2 = 0.890. This method has a detection limit of 0.00004 uM 5-AZA and good repeatability with variation of 7.5%-23.9%. Almost half of the aquatic from the Tianjin basin was found demethylation ability positively with this method.