摘要
目的：观察人胚肾细胞（HEK293）短期暴露于高剂量氯化镉（CdCl2）时，TET酶表达及DNA甲基化水平的变化。方法 HEK293细胞经CdCl2染毒24、48和72 h后，用细胞增殖与活性检测试剂盒（CCK-8）检测细胞存活率并记录细胞形态，Real-time PCR法检测mRNA表达水平，Western blot法检测TET3酶蛋白表达水平，并采用焦磷酸测序法检测全基因组DNA甲基化水平。结果细胞增殖及细胞存活率监测结果显示，CdCl2对HKE293细胞有毒性作用，其半数抑制浓度（IC50）为1.78μmol/L；CdCl2处理HEK293细胞24、48和72 h后，细胞形态发生改变，2.0μmol/L染毒组细胞呈空泡样变化且形态模糊。染毒剂量分别为0.0、0.5、1.0和2.0μmol/L时，TET1 mRNA相对表达量分别为0.23±0.13、0.48±0.12、0.59±0.16和0.95±0.39（F=182.89，P=0.002）；TET2 mRNA相对表达量分别为0.23±0.12、0.32±0.02、0.31±0.10和0.34±0.07（F=18.91，P〈0.001）；TET3 mRNA相对表达量分别为0.26±0.10、0.27±0.11、0.25±0.11和0.28±0.09（F=1.76，P=0.036）。TET1 mRNA、TET2 mRNA和TET3 mRNA相对表达量在染毒时间和染毒剂量均有交互作用（F值分别为32.94、23.04和13.78，P值分别为〈0.001、0.041和〈0.001）。Western blot检测结果显示，TET酶蛋白表达量在不同时间梯度和浓度梯度的改变趋势与mRNA相对表达量一致。染毒时间分别为24、48和72 h时，全基因组DNA甲基化水平分别为（55.01±3.62）%、（48.31±8.99）%、（48.76±6.60）%（F=18.50，P〈0.001）；染毒剂量分别为0.0、0.5、1.0和2.0μmol/L时，全基因组DNA甲基化水平分别为（55.29±2.83）%、（55.35±3.11）%、（48.58±6.40）%和（43.56±7.89）%（F=7.03，P=0.048）。全基因组DNA甲基化水平在剂量组和时间点有交互作用（F=2.73， P=0.043）。结论在CdCl2短期暴露下，随着染毒剂量和染毒时间的增加，TET酶mRNA和蛋白表达水平呈逐渐升高的趋势，且全基因组DNA也表现出去甲
Objective To detect the expression changes of the demethylase TETs （Ten-eleven translocation enzymes） in human embryonic kidney cell （HEK293） exposed to high dose cadmium chloride （CdCl2）, and to investigate the regulation effects of TETs on global genomic methylation. Methods HEK293 cells were exposed to CdCl2 for 24 h, 48 h and 72 h, the survival rate was tested by CCK-8 （cell counting kit-8） method, and the cell morphology was observed. The levels of TETs mRNA and protein were detected by fluorescence quantitative PCR and Western blot, respectively. The genomic DNA methylation level was detectedby pyro sequencing assay. Results CdCl2 had toxic effects on HEK293 cells, and the half inhibitory concentration （IC50） was 1.78 μmol/L. After exposure of CdCl2 for 24 h, 48 h and 72 h, the morphology of HEK293 cells was altered, and the high dose group （2.0 μmol/L） showed vacuolar changes 〈br〉 and fuzzy appearance. The level of TET1 mRNA in groups of 0.0,0.5,1.0,2.0μmol/L were 0.23±0.13,0.48± 0.12,0.59±0.16 and 0.95±0.39, respectively （F=182.89,P=0.002）;The level of TET2 mRNA in groups of 0.0, 0.5,1.0,2.0μmol/L were 0.23±0.12,0.32±0.02,0.31±0.10 and 0.34±0.07, respectively （F=27.94,P〈0.001）;The level of TET3 mRNA in groups of 0.0,0.5,1.0,2.0 μmol/L were 0.26 ± 0.10,0.27 ± 0.11,0.25 ± 0.11 and 0.28 ± 0.09, respectively （F=1.76,P=0.036）.The interaction effect existed between exposure time and doses of TET1 mRNA,TET2 mRNA and TET3 mRNA （F values were 32.94,23.04 and 13.78,respectively;P values were 〈0.001,0.041 and 〈0.001,respectively）.Western blot showed that in different exposure time and dose, the protein expression levels of TETs had the similar trend as mRNA levels.In 24 h （55.01 ± 3.62）%,48 h （48.31±8.99）%, 72 h （48.76±6.60）%, the DNA methylation had significant differences（F=18.50,P〈0.001）;In groups of 0.0 μmol/L（55.29±2.83）%,0.5 μmol/L（55.35±3.11）%,1.0 μmol/L（48.58±6.40）% and 2.0 μmol/L（43.56 ± 7.89）%, t
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2015年第9期822-827,共6页
Chinese Journal of Preventive Medicine
基金
国家自然科学基金（81101562、81273099）
中央高校基本科研基金（12ykpy13）
广东省自然科学基金（s2012010009633）
广东省科技计划（2012b060300005）
广州市卫生局重点学科建设（2013-2015-07）
