本文選題：抗結(jié)核藥物 + 肝損傷��； 參考：《華北理工大學(xué)》2017年碩士論文

【摘要】：目的研究抗結(jié)核藥物性肝損傷(anti-tuberculosis drug-induced liver injury,ADLI)中環(huán)狀RNA(circular RNA,circRNA)的表達差異,尋找與ADLI有關(guān)的circRNA。方法主要從細胞和人群兩方面進行研究。細胞研究中,將不同濃度的兩藥聯(lián)合(異煙肼+利福平)、三藥聯(lián)合(異煙肼+利福平+吡嗪酰胺)藥物作用于HL-7702人正常肝細胞系,CCK8法檢測加藥48h后細胞存活率,篩選出最佳濃度(存活率70%-80%)。以最佳濃度建立肝細胞損傷模型,分為兩藥聯(lián)合組、三藥聯(lián)合組和正常對照組,各組分別在加藥3、6、12、24、36、48h后收集細胞和細胞培養(yǎng)上清液。ALT、AST的檢測利用賴氏法進行;常規(guī)Trizol法提取肝細胞的總RNA;芯片技術(shù)篩選差異表達circRNA;miRanda-3.3軟件預(yù)測circRNA的靶miRNA;RTqPCR法驗證差異表達circRNA。單因素方差分析或t檢驗判斷ALT、AST和circRNA在組間是否有差異,Pearson相關(guān)分析判斷circRNA與ALT、AST之間是否相關(guān)。人群研究中,收集2015年7月~2016年7月在唐山結(jié)核病院被確診為肺結(jié)核的住院患者的臨床資料和血標(biāo)本。將符合條件的病例分別納入肝損傷組和非肝損傷組,用于差異表達circRNA的篩選(每組16例)和驗證(每組98例),非肝損傷組以抗結(jié)核藥物化療方案、性別、年齡為匹配因素,與肝損傷組進行1:1匹配。circRNA的篩選、驗證和靶miRNA預(yù)測方法同HL-7702肝細胞實驗。利用配對t檢驗或配對χ2檢驗判斷人群資料、circRNA、ALT和AST是否具有組間差異;利用Pearson相關(guān)分析判斷circRNA與ALT、AST之間是否相關(guān)。結(jié)果兩藥聯(lián)合誘導(dǎo)肝細胞損傷模型組中差異表達的circRNA共40965個,其中表達上調(diào)的circRNA共14863個,表達下調(diào)的circRNA 26102個。三藥聯(lián)合誘導(dǎo)肝細胞損傷模型組中差異表達的circRNA共10248個,其中表達上調(diào)的circRNA共2320個,表達下調(diào)的circRNA共7928個。ADLI患者血漿中差異表達的circRNA共6661個,其中表達上調(diào)的circRNA共273個,表達下調(diào)的circRNA共6388個。在兩藥和三藥聯(lián)合肝細胞損傷模型以及ADLI患者血漿中均差異表達的circRNA共112個;其中在兩藥組和ADLI患者血漿中均上調(diào)倍數(shù)最大的是hsa_circ_0010996,在三藥組中上調(diào)倍數(shù)最大的是hsa_circ_0033188;分別在兩藥組、三藥組和ADLI患者血漿中下調(diào)倍數(shù)最大hsa_circ_0012054、hsa_circ_0025088和hsa_circ_0021214。差異表達circRNA靶mi RNA預(yù)測結(jié)果顯示,除hsa_circ_0021214外只預(yù)測到92個靶miRNA外其余4個circRNA均預(yù)測到100個靶miRNA。差異表達circRNA驗證結(jié)果顯示,hsa_circ_0010996在兩藥組、三藥組和ADLI患者血漿中均表達上調(diào),與芯片篩選結(jié)果一致。結(jié)論在兩藥聯(lián)合和三藥聯(lián)合誘導(dǎo)的肝細胞損傷模型中分別篩選到40965個和10248個circRNA,患者血漿中篩選到6661個circRNA。細胞中與血漿中差異表達一致的circRNA有112個。得到驗證的circRNA是hsa_circ_0010996,與肝功指標(biāo)ALT和AST具有相關(guān)性,說明其與ADLI密切相關(guān)。
[Abstract]:Objective to study the expression of cyclic drug-induced RNA (RNA(circular) in anti-tuberculosis drug-induced liver injury-induced liver injury (ADLI) and to find the ADLI related circRNAs.Methods two aspects of cell and population were studied.In cell study, the cell survival rate of HL-7702 human normal liver cell line was measured by CCK8 method with two different concentrations (isoniazid rifampicin) and three drugs (isoniazid rifampicin pyrazinamide).Select the best concentration (survival rate 70-80).The model of hepatocyte injury was established at the best concentration, and was divided into two groups: the combined group, the combined group and the control group. The cells and the supernatant of cell culture were collected for 48 hours after the addition of 3 drugs. The detection of the supernatant of cell and cell culture was carried out by the method of Lai's method.The total RNAs of hepatocytes were extracted by conventional Trizol method, and the differential expression of circRNA-miRanda-3.3 was screened by microarray technique, and the differential expression of circRNA was verified by RT-PCR with target miRNA-RT qPCR for predicting circRNA.Univariate ANOVA or t-test was used to determine whether there was any difference between circRNA and alt. Pearson correlation analysis was used to determine the correlation between circRNA and alt.From July 2015 to July 2016, clinical data and blood samples of inpatients diagnosed with tuberculosis in Tangshan Tuberculosis Hospital were collected.The eligible cases were included in the liver injury group and the non-hepatic injury group, respectively, and were used to screen for differential expression of circRNA (16 cases in each group) and to verify (98 cases per group). The non-hepatic injury group was matched by anti-tuberculosis drug chemotherapy regimen, sex and age.The 1:1 matched. CircRNA was screened with liver injury group, and the method of target miRNA prediction was same as that of HL-7702 hepatocyte experiment.Paired t test or paired 蠂 2 test were used to determine whether there was a difference between alt and AST in crowd data, and Pearson correlation analysis was used to determine the correlation between circRNA and alt.Results there were 40965 differentially expressed circRNA in the model group of hepatocyte injury induced by two drugs, including 14863 up-regulated circRNA and 26102 down-regulated circRNA.A total of 10248 circRNA were differentially expressed in the model group of hepatocyte injury induced by three drugs, of which 2,320 were up-regulated circRNA, and 6661 were differentially expressed circRNA in 7928. ADLI patients with down-regulated circRNA, 273 of which were up-regulated circRNA.The expression of down-regulated circRNA was 6388.There were 112 differentially expressed circRNA in plasma of patients with ADLI and two drugs and three drugs combined with hepatocyte injury model.In the two drugs group and ADLI group, the biggest up-regulation was hsacirccirctig _ 0 096, and in the three-drug group, the biggest up-regulation was hsacirccirc000033188; in the two drug groups, the largest down-regulation multiple was hsacirccirccirccirc0025088 and hsacirccirccirccirc0025088 and hsacirccirccirccirc0025088 and hsacirc0021214in the two drugs group, three drug group and ADLI group respectively.The predicted results of differentially expressed circRNA target mi RNA showed that all the 4 circRNA except hsa_circ_0021214 predicted only 92 target miRNA and 100 target miRNAs were predicted.The results of differential expression circRNA test showed that the expression of hsa-tid circ0010996 was up-regulated in plasma of two drug groups, three drug groups and ADLI patients, which was consistent with the results of microarray screening.Conclusion 40965 circRNAs and 10248 circRNAs were screened in the hepatocyte injury model induced by the combination of two drugs and three drugs, and 6661 circRNAs were screened in the plasma of the patients.There were 112 circRNA differentially expressed in cell and plasma.The verified circRNA was hsac _ s _ 0 ~ 10996, which was correlated with liver function index ALT and AST, indicating that it was closely related to ADLI.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R52;R575

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本文編號：1760757