【摘要】：目的探討CYP1A1啟動(dòng)子區(qū)CpG島甲基化與抗結(jié)核藥物性肝損傷(antituberculosis drug-induced hepatic injury,ADIH)的關(guān)系以及5-氮雜-2'-脫氧胞苷(5-aza-2'-deoxycytidine,5-Aza-Cd R)對(duì)ADIH的作用。方法將HL-7702細(xì)胞分為對(duì)照組、異煙肼組(INH)、利福平組(RFP)、吡嗪酰胺組(PZA)、兩藥聯(lián)合組(INH+RFP)、三藥聯(lián)合組(INH+RFP+PZA)。首先,以不同濃度的抗結(jié)核藥作用肝細(xì)胞24h,采用CCK8法檢測(cè)各組藥物對(duì)細(xì)胞存活率的影響,來(lái)確定不同種類抗結(jié)核藥的濃度。其次,依據(jù)上述確定的各組藥物濃度處理肝細(xì)胞,均培養(yǎng)6h、12h、24h,賴氏法檢測(cè)不同藥物組各時(shí)間點(diǎn)LDH、SOD、MDA的變化;以熒光PCR(q RT-PCR)法檢測(cè)CYP1A1 m RNA表達(dá)變化,并將CYP1A1 m RNA與LDH、SOD、MDA水平進(jìn)行相關(guān)性分析;選取CYP1A1 m RNA變化最大時(shí)間點(diǎn),采用甲基化測(cè)序法檢測(cè)不同抗結(jié)核藥處理后CYP1A1啟動(dòng)子區(qū)CpG島甲基化情況。最后,采用5-Aza-Cd R干預(yù)肝細(xì)胞后,以熒光PCR(q RT-PCR)法檢測(cè)CYP1A1和DNA甲基轉(zhuǎn)移酶(DNMT1、DNMT3a、DNMT3b)m RNA表達(dá),MSP法檢測(cè)不同種類抗結(jié)核藥CYP1A1啟動(dòng)子區(qū)CpG島甲基化狀態(tài),ELISA檢測(cè)CYP1A1蛋白、DNMT1、DNMT3a、DNMT3b蛋白和酶活性的變化,賴氏法檢測(cè)法LDH、SOD、MDA水平變化。結(jié)果依據(jù)細(xì)胞存活率,確定了各組藥物濃度分別為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)。用上述各藥物濃度培養(yǎng)細(xì)胞6h、12h、24h后,發(fā)現(xiàn)反映肝細(xì)胞損傷的指標(biāo)LDH、SOD、MDA均發(fā)生異常改變,各藥物組LDH、MDA明顯上升,SOD顯著下降,均在24h達(dá)到最大變化(P0.01),且三藥聯(lián)合組的變化幅度最大(P0.01),提示不同種類抗結(jié)核藥均可導(dǎo)致肝細(xì)胞損傷,且三藥聯(lián)合損傷更重。細(xì)胞經(jīng)INH、PZA、兩藥聯(lián)合、三藥聯(lián)合處理后,CYP1A1 m RNA表達(dá)水平均呈先下降后回升的趨勢(shì),均在12h下降到最低值(均P0.05),且三藥聯(lián)合下降幅度最大;而RFP處理后是呈先升高后下降趨勢(shì),且24h達(dá)到最低值;以上結(jié)果提示,INH、PZA、兩藥聯(lián)合、三藥聯(lián)合致肝損傷過(guò)程中,CYP1A1發(fā)生低表達(dá)。CYP1A1的低表達(dá)可能受啟動(dòng)子區(qū)甲基化調(diào)控,進(jìn)一步對(duì)各類藥物CYP1A1啟動(dòng)子區(qū)CpG島甲基化測(cè)序,結(jié)果表明,INH、PZA、兩藥聯(lián)合、三藥聯(lián)合組CYP1A1啟動(dòng)子區(qū)甲基化率均升高(均P0.05),三藥聯(lián)合組甲基化率高于兩藥聯(lián)合組;RFP組甲基化率雖升高,但無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),以上結(jié)果提示,INH、PZA、兩藥聯(lián)合、三藥聯(lián)合致肝細(xì)胞損傷中,CYP1A1的低表達(dá)可能受其啟動(dòng)子區(qū)高甲基化調(diào)控。針對(duì)上升各組的甲基化變化,觀察加入甲基化抑制劑5-Aza-Cd R處理后的效果;MSP結(jié)果顯示,加入抑制劑后,INH、PZA、兩藥聯(lián)合、三藥聯(lián)合組CYP1A1啟動(dòng)子區(qū)CpG島甲基化率降低,且三藥聯(lián)合組降低最顯著。同時(shí),CYP1A1蛋白和m RNA表達(dá)水平均升高,蛋白比m RNA表達(dá)水平升高更明顯,且三藥聯(lián)合組加入抑制劑后CYP1A1 m RNA及蛋白的表達(dá)變化最顯著(P0.01),提示5-Aza-Cd R抑制CYP1A1基因甲基化促進(jìn)其表達(dá)。結(jié)論CYP1A1啟動(dòng)子區(qū)CpG島高甲基化調(diào)控CYP1A1低表達(dá),可能與抗結(jié)核藥物致肝細(xì)胞損傷有關(guān)。5-Aza-Cd R可通過(guò)去甲基化作用升高CYP1A1表達(dá),在一定程度上緩解藥物性肝細(xì)胞損傷。
[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.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575;R52

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相關(guān)期刊論文 前1條

1 李艷輝;李玉紅;朱凌妍;王吉順;穆莎莎;馮福民;;異煙肼導(dǎo)致大鼠肝損傷后對(duì)基因啟動(dòng)子區(qū)CpG島甲基化水平的影響[J];中國(guó)臨床藥理學(xué)雜志;2017年02期

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本文編號(hào)：2181840