二甲苯体外诱导肾小管上皮细胞损伤的表观遗传机制

发布时间：2018-12-19 14:54

【摘要】：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,P0.01]。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。
[Abstract]:Aim: to study the characteristics and epigenetic mechanism of renal tubular epithelial cell injury induced by xylene. Methods: human renal tubular epithelial cells (HK-2) were treated with different concentrations of xylene in vitro. The expression of neutrophil gelatinase-associated lipid carrier protein (NGAL) in supernatant of cultured human renal tubular epithelial cells (TECs) was detected by ELISA assay. Apoptosis of renal tubular epithelial cells was detected by flow cytometry and Caspase-3 activity was detected by Caspase-3 assay kit. Methylation screening and verification of the CG site of the DNA of renal tubular epithelial cells induced by xylene were performed by human methylation 450K chip and pyrosequencing. Results: the expression of NGAL in HK-2 cells was increased in a dose-and time-dependent manner. The value of NGAL in supernatant cultured with 1.4mmol/L xylene for 48 h was significantly higher than that in control group [(4.52 卤0.49) pg/ml vs (2.30 卤0.11) pg/ml,P0.01]. With the increase of the concentration of xylene, the ratio of apoptosis and the activity of Caspase-3 increased significantly in HK-2 cells. The results of methylation chip analysis showed that there were significant differences in methylation of HK-2 cells induced by xylene, of which 109 showed increased methylation level and 134 showed decreased methylation level. A total of 138 genes were involved. Among them, apoptosis-related genes (such as Bax,FAF1) showed significant methylation changes, FAF1 methylation level decreased, and Bax methylation level increased. The results of pyrosequencing showed that the methylation level of Bax gene (92%) was 7% higher than that of control group (85%). The methylation level of FAF1 gene was 6% lower than that of control group, and the trend of methylation change was consistent with the results of microarray. Conclusion: the methylation level of apoptosis-related genes such as Bax,FAF1 may be involved in the injury of renal tubular epithelial cells induced by xylene, and the mechanism needs further study.
【作者单位】：南京大学医学院附属金陵医院(南京军区南京总医院)肾脏科国家肾脏疾病临床医学研究中心全军肾脏病研究所;
【基金】：国家重点基础研究发展计划[973计划(2012CB517600)(2012CB517605)] 国家自然科学基金(81270811)
【分类号】：R114

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