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 and epigenetic mechanism of xylene induced renal tubular epithelial cell injury. Methods: Different concentrations of xylene were applied to human renal tubular epithelial cells (HK-2) cultured in vitro. The expression of neutrophil gelatinase-associated lipocalin (NGAL) in renal tubular epithelial cell culture supernatant was detected by ELISA. The expression of Caspase-3 was detected by flow cytometry. Caspase-3 activity was detected by Caspase-3 activity assay kit. Methylation screening and validation of the CG sites of xylene-induced tubular epithelial cells were performed using human methylated 450K chip and pyrosequencing. Results: The expression of NGAL in HK-2 cells induced by xylene increased dose-dependently and time-dependently. Compared with the control group, the level of NGAL in the supernatant of the culture supernatant after 1.4 mmol / L xylene treatment increased significantly [(4.52 ± 0.49) pg / ml vs (2.30 ± 0.11) pg / ml, P <0.01]. With the increase of xylene concentration, the percentage of apoptosis and the activity of Caspase-3 in HK-2 cells increased significantly. The results of methylation analysis showed that methylation of 243 probes in HK-2 cells was significantly different after xylene stimulation. Among them, 109 probes showed higher methylation level and 134 probes showed methyl Reduced levels, a total of 138 genes involved. Among them, the apoptosis-related genes (such as Bax, FAF1, etc.) showed a significant change in methylation, FAF1 methylation decreased, Bax methylation increased. Pyrosequencing analysis showed that the methylation level of Bax gene (92%) was 7% higher than that of the control group (85%) after xylene stimulation. The methylation level of FAF1 gene was higher than that of the control Group decreased by 6%, the trend of its methylation changes consistent with the results of the chip. CONCLUSION: Xylene stimulation can induce the injury and apoptosis of HK-2 cells. The methylation level of Bax, FAF1 and other apoptosis-related genes may be involved in the damage of renal tubular epithelial cells induced by xylene. The mechanism remains to be further studied.