二甲苯体外诱导肾小管上皮细胞损伤的表观遗传机制

Epigenetic mechanisms of xylene-induced renal tubular epithelial cell injury

目的:研究二甲苯诱导肾小管上皮细胞损伤的特点及表观遗传机制。方法:不同浓度的二甲苯分别作用于体外培养的人肾小管上皮细胞(HK-2),通过ELISA法检测肾小管上皮细胞培养上清中中性粒细胞明胶酶相关脂质运载蛋白(NGAL)表达变化,流式技术检测肾小管上皮细胞凋亡,Caspase-3活性检测试剂盒检测Caspase-3活性。采用人甲基化450K芯片和焦磷酸测序对二甲苯诱导损伤的肾小管上皮细胞DNA的CG位点进行甲基化筛选和验证。结果:二甲苯诱导刺激后HK-2细胞NGAL表达增加,呈现剂量和时间依赖性;1.4mmol/L二甲苯作用48h后培养上清中NGAL的值较对照组显著增加[(4.52±0.49)pg/ml vs(2.30±0.11)pg/ml,P<0.01]。随加入二甲苯浓度的增加,HK-2细胞的凋亡比例和Caspase-3活性明显增加。甲基化芯片分析结果显示,二甲苯刺激后,HK-2细胞有243条探针甲基化出现显著差异,其中109条探针显示为甲基化水平增高,134条探针显示为甲基化水平降低,共涉及到138个基因。其中,细胞凋亡相关的基因(如Bax、FAF1等)出现了明显的甲基化改变,FAF1甲基化水平降低,Bax甲基化水平升高。焦磷酸测序的分析结果显示,二甲苯刺激后Bax基因(92%)的甲基化水平与对照组(85%)相比出现7%的甲基化改变;FAF1基因的甲基化水平比对照组降低6%,其甲基化改变的趋势与芯片结果相一致。结论:二甲苯刺激可引起HK-2细胞的损伤和凋亡增加,Bax、FAF1等凋亡相关基因的甲基化水平改变可能参与二甲苯对肾小管上皮细胞损伤,其机制有待进一步研究。

Objective： To investigate the characteristics of proximal tubule epithelial cells injury and the epigenetic mechanism of the injury induced by organic solvents. Methodology： Human proximal tubule epithelial cells were treated with different concentrations of xylene. The NGAL in the culture medium was measured by ELISA, the apoptosis of tubular ceils was detected by flow cytometry and the Caspase-3 activity was detected by kit. The Illumina Infinium Human Methylation 450 （450K） Bead Chips were used to detect the methylation changes in HK-2 cells after xylene treatment. The data of Illumina chips were validated by pyro-sequencing method. Results：The secretion of NGAL was increased in dose-and time-dependent manner after xylene treatment. The NGAL was increased significantly [ （4. 52± 0. 49） pg/ml vs （2. 30±0. 11） pg/ml, P〈0. 013 after 48 h treatment with 1.4 mmo]/L xylene. At the same time, with increasing concentrations of xylene, the ratio of HK-2 cells apoptosis and Caspase-3 activity was significantly increased. Xylene treatment induced DNA methylation changes, indicating by 243 probes in HK-2 cells after xylene stimulation, with 109 probes displayed increased levels of methylation and 134 probes showed reduced levels of methylation. The methylation changes of 138 genes were indicated by these probes, including apoptosis related gene, e.g. Bax, FAF1, etc. The DNA methylation of FAF1 was decreased while the DNA methylation of BAX was increased. The pyrosequencing assays showed that Bax gene methylation level changes of xylene stimulation group was 7% compared to control group, and FAF1 gene was 6%, its methylation change trend was consistent with the results of the chip. Conclusion： Xylene stimulation can cause HK-2 cells injury and increased apoptosis. The changes of DNA methylation level of Bax, FAF1 might involve in xylene induced injury.