CYP1A1启动子区甲基化在抗结核药物致肝细胞损伤中的作用
发布时间：2018-08-13 18:38
【摘要】：目的探讨CYP1A1启动子区CpG岛甲基化与抗结核药物性肝损伤(antituberculosis drug-induced hepatic injury,ADIH)的关系以及5-氮杂-2'-脱氧胞苷(5-aza-2'-deoxycytidine,5-Aza-Cd R)对ADIH的作用。方法将HL-7702细胞分为对照组、异烟肼组(INH)、利福平组(RFP)、吡嗪酰胺组(PZA)、两药联合组(INH+RFP)、三药联合组(INH+RFP+PZA)。首先,以不同浓度的抗结核药作用肝细胞24h,采用CCK8法检测各组药物对细胞存活率的影响,来确定不同种类抗结核药的浓度。其次,依据上述确定的各组药物浓度处理肝细胞,均培养6h、12h、24h,赖氏法检测不同药物组各时间点LDH、SOD、MDA的变化;以荧光PCR(q RT-PCR)法检测CYP1A1 m RNA表达变化,并将CYP1A1 m RNA与LDH、SOD、MDA水平进行相关性分析;选取CYP1A1 m RNA变化最大时间点,采用甲基化测序法检测不同抗结核药处理后CYP1A1启动子区CpG岛甲基化情况。最后,采用5-Aza-Cd R干预肝细胞后,以荧光PCR(q RT-PCR)法检测CYP1A1和DNA甲基转移酶(DNMT1、DNMT3a、DNMT3b)m RNA表达,MSP法检测不同种类抗结核药CYP1A1启动子区CpG岛甲基化状态,ELISA检测CYP1A1蛋白、DNMT1、DNMT3a、DNMT3b蛋白和酶活性的变化,赖氏法检测法LDH、SOD、MDA水平变化。结果依据细胞存活率,确定了各组药物浓度分别为800μg/ml INH、300μg/ml RFP、500μg/ml PZA、50μg/ml+100μg/ml(INH+RFP)、85.5μg/ml+427.5μg/ml+171μg/ml(INH+RFP+PZA)。用上述各药物浓度培养细胞6h、12h、24h后,发现反映肝细胞损伤的指标LDH、SOD、MDA均发生异常改变,各药物组LDH、MDA明显上升,SOD显著下降,均在24h达到最大变化(P0.01),且三药联合组的变化幅度最大(P0.01),提示不同种类抗结核药均可导致肝细胞损伤,且三药联合损伤更重。细胞经INH、PZA、两药联合、三药联合处理后,CYP1A1 m RNA表达水平均呈先下降后回升的趋势,均在12h下降到最低值(均P0.05),且三药联合下降幅度最大;而RFP处理后是呈先升高后下降趋势,且24h达到最低值;以上结果提示,INH、PZA、两药联合、三药联合致肝损伤过程中,CYP1A1发生低表达。CYP1A1的低表达可能受启动子区甲基化调控,进一步对各类药物CYP1A1启动子区CpG岛甲基化测序,结果表明,INH、PZA、两药联合、三药联合组CYP1A1启动子区甲基化率均升高(均P0.05),三药联合组甲基化率高于两药联合组;RFP组甲基化率虽升高,但无统计学意义(P0.05),以上结果提示,INH、PZA、两药联合、三药联合致肝细胞损伤中,CYP1A1的低表达可能受其启动子区高甲基化调控。针对上升各组的甲基化变化,观察加入甲基化抑制剂5-Aza-Cd R处理后的效果;MSP结果显示,加入抑制剂后,INH、PZA、两药联合、三药联合组CYP1A1启动子区CpG岛甲基化率降低,且三药联合组降低最显著。同时,CYP1A1蛋白和m RNA表达水平均升高,蛋白比m RNA表达水平升高更明显,且三药联合组加入抑制剂后CYP1A1 m RNA及蛋白的表达变化最显著(P0.01),提示5-Aza-Cd R抑制CYP1A1基因甲基化促进其表达。结论CYP1A1启动子区CpG岛高甲基化调控CYP1A1低表达,可能与抗结核药物致肝细胞损伤有关。5-Aza-Cd R可通过去甲基化作用升高CYP1A1表达,在一定程度上缓解药物性肝细胞损伤。
[Abstract]:Objective To investigate the relationship between CpG island methylation in promoter region of CYP1A1 and anti-tuberculosis drug-induced hepatic injury (ADIH) and the effect of 5-aza-2'-deoxycytidine (5-Aza-Cd R) on ADIH. Methods HL-7702 cells were divided into control group, isoniazid group (INH), rifampicin group (RFP), pyrazine group. Amide group (PZA), two-drug combination group (INH+RFP), three-drug combination group (INH+RFP+PZA). Firstly, liver cells were treated with different concentrations of anti-tuberculosis drugs for 24 hours. CCK8 method was used to detect the effect of each group of drugs on cell survival rate to determine the concentration of different kinds of anti-tuberculosis drugs. Secondly, liver cells were treated with the above-mentioned concentrations of drugs and cultured for 6 hours. The changes of LDH, SOD and MDA in different drug groups at different time points were detected by Rye's method; the expression of CYP1A1 m RNA was detected by fluorescence PCR (q RT-PCR), and the correlation between CYP1A1 m RNA and the levels of LDH, SOD and MDA was analyzed; the maximum time point of change of CYP1A1 m RNA was selected, and the start-up of CYP1A1 was detected by methylation sequencing. Finally, after 5-Aza-Cd R intervention, the expression of CYP1A1 and DNA methyltransferase (DNMT1, DNMT3a, DNMT3b) m RNA were detected by fluorescence PCR (q RT-PCR), the methylation status of CpG island in promoter region of different anti-tuberculosis drugs CYP1A1 was detected by MSP, and the activities of CYP1A1, DNMT1, DNMT3a, DNMT3b proteins and enzymes were detected by ELISA. Results According to the cell survival rate, the drug concentrations of each group were determined as 800 ug/ml INH, 300 ug/ml RFP, 500 ug/ml PZA, 50 ug/ml+100 ug/ml (INH+RFP), 85.5 ug/ml+427.5 ug/ml+171 ug/ml (INH+RFP+PZA). The cells were cultured for 6 hours, 12 hours and 24 hours, respectively. LDH, SOD, MDA were abnormal changes, LDH, MDA increased significantly, SOD decreased significantly in each drug group, and reached the maximum change in 24 hours (P The expression of CYP1A1 m RNA decreased at first and then increased after treatment with three drugs, and all decreased to the lowest level at 12 hours (all P 0.05), and the greatest decrease was observed after treatment with three drugs; however, after treatment with RFP, the expression of CYP1 m RNA increased first and then decreased, and reached the lowest level at 24 hours; the above results suggest that INH, PZA, two drugs combined with three drugs can cause liver injury. Low expression of CYP1A1 may be regulated by promoter methylation. Further methylation of CpG island in promoter region of CYP1A1 was sequenced. The results showed that the methylation rate of promoter region of CYP1A1 in INH, PZA, combination of the two drugs, combination of the three drugs group increased (all P 0.05), and the methylation rate in combination group was higher than that in combination group. These results suggest that the low expression of CYP1A1 may be regulated by hypermethylation of its promoter region in hepatocyte injury induced by INH, PZA, combination of two drugs and three drugs. The methylation rate of CpG island in the promoter region of CYP1A1 in the three-drug combination group was decreased, and the most significant decrease was found in the three-drug combination group. Conclusion CpG island hypermethylation in the promoter region of CYP1A1 may regulate the low expression of CYP1A1, which may be related to the hepatocyte injury induced by anti-tuberculosis drugs.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575;R52
本文编号：2181840
[Abstract]:Objective To investigate the relationship between CpG island methylation in promoter region of CYP1A1 and anti-tuberculosis drug-induced hepatic injury (ADIH) and the effect of 5-aza-2'-deoxycytidine (5-Aza-Cd R) on ADIH. Methods HL-7702 cells were divided into control group, isoniazid group (INH), rifampicin group (RFP), pyrazine group. Amide group (PZA), two-drug combination group (INH+RFP), three-drug combination group (INH+RFP+PZA). Firstly, liver cells were treated with different concentrations of anti-tuberculosis drugs for 24 hours. CCK8 method was used to detect the effect of each group of drugs on cell survival rate to determine the concentration of different kinds of anti-tuberculosis drugs. Secondly, liver cells were treated with the above-mentioned concentrations of drugs and cultured for 6 hours. The changes of LDH, SOD and MDA in different drug groups at different time points were detected by Rye's method; the expression of CYP1A1 m RNA was detected by fluorescence PCR (q RT-PCR), and the correlation between CYP1A1 m RNA and the levels of LDH, SOD and MDA was analyzed; the maximum time point of change of CYP1A1 m RNA was selected, and the start-up of CYP1A1 was detected by methylation sequencing. Finally, after 5-Aza-Cd R intervention, the expression of CYP1A1 and DNA methyltransferase (DNMT1, DNMT3a, DNMT3b) m RNA were detected by fluorescence PCR (q RT-PCR), the methylation status of CpG island in promoter region of different anti-tuberculosis drugs CYP1A1 was detected by MSP, and the activities of CYP1A1, DNMT1, DNMT3a, DNMT3b proteins and enzymes were detected by ELISA. Results According to the cell survival rate, the drug concentrations of each group were determined as 800 ug/ml INH, 300 ug/ml RFP, 500 ug/ml PZA, 50 ug/ml+100 ug/ml (INH+RFP), 85.5 ug/ml+427.5 ug/ml+171 ug/ml (INH+RFP+PZA). The cells were cultured for 6 hours, 12 hours and 24 hours, respectively. LDH, SOD, MDA were abnormal changes, LDH, MDA increased significantly, SOD decreased significantly in each drug group, and reached the maximum change in 24 hours (P The expression of CYP1A1 m RNA decreased at first and then increased after treatment with three drugs, and all decreased to the lowest level at 12 hours (all P 0.05), and the greatest decrease was observed after treatment with three drugs; however, after treatment with RFP, the expression of CYP1 m RNA increased first and then decreased, and reached the lowest level at 24 hours; the above results suggest that INH, PZA, two drugs combined with three drugs can cause liver injury. Low expression of CYP1A1 may be regulated by promoter methylation. Further methylation of CpG island in promoter region of CYP1A1 was sequenced. The results showed that the methylation rate of promoter region of CYP1A1 in INH, PZA, combination of the two drugs, combination of the three drugs group increased (all P 0.05), and the methylation rate in combination group was higher than that in combination group. These results suggest that the low expression of CYP1A1 may be regulated by hypermethylation of its promoter region in hepatocyte injury induced by INH, PZA, combination of two drugs and three drugs. The methylation rate of CpG island in the promoter region of CYP1A1 in the three-drug combination group was decreased, and the most significant decrease was found in the three-drug combination group. Conclusion CpG island hypermethylation in the promoter region of CYP1A1 may regulate the low expression of CYP1A1, which may be related to the hepatocyte injury induced by anti-tuberculosis drugs.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575;R52
【参考文献】
相关期刊论文 前1条
1 李艳辉;李玉红;朱凌妍;王吉顺;穆莎莎;冯福民;;异烟肼导致大鼠肝损伤后对基因启动子区CpG岛甲基化水平的影响[J];中国临床药理学杂志;2017年02期
，本文编号：2181840
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