抗结核药物性肝损伤中环状RNA表达差异研究

发布时间：2018-04-16 22:06

  本文选题：抗结核药物 + 肝损伤　； 参考：《华北理工大学》2017年硕士论文

【摘要】：目的研究抗结核药物性肝损伤(anti-tuberculosis drug-induced liver injury,ADLI)中环状RNA(circular RNA,circRNA)的表达差异,寻找与ADLI有关的circRNA。方法主要从细胞和人群两方面进行研究。细胞研究中,将不同浓度的两药联合(异烟肼+利福平)、三药联合(异烟肼+利福平+吡嗪酰胺)药物作用于HL-7702人正常肝细胞系,CCK8法检测加药48h后细胞存活率,筛选出最佳浓度(存活率70%-80%)。以最佳浓度建立肝细胞损伤模型,分为两药联合组、三药联合组和正常对照组,各组分别在加药3、6、12、24、36、48h后收集细胞和细胞培养上清液。ALT、AST的检测利用赖氏法进行;常规Trizol法提取肝细胞的总RNA;芯片技术筛选差异表达circRNA;miRanda-3.3软件预测circRNA的靶miRNA;RTqPCR法验证差异表达circRNA。单因素方差分析或t检验判断ALT、AST和circRNA在组间是否有差异,Pearson相关分析判断circRNA与ALT、AST之间是否相关。人群研究中,收集2015年7月~2016年7月在唐山结核病院被确诊为肺结核的住院患者的临床资料和血标本。将符合条件的病例分别纳入肝损伤组和非肝损伤组,用于差异表达circRNA的筛选(每组16例)和验证(每组98例),非肝损伤组以抗结核药物化疗方案、性别、年龄为匹配因素,与肝损伤组进行1:1匹配。circRNA的筛选、验证和靶miRNA预测方法同HL-7702肝细胞实验。利用配对t检验或配对χ2检验判断人群资料、circRNA、ALT和AST是否具有组间差异;利用Pearson相关分析判断circRNA与ALT、AST之间是否相关。结果两药联合诱导肝细胞损伤模型组中差异表达的circRNA共40965个,其中表达上调的circRNA共14863个,表达下调的circRNA 26102个。三药联合诱导肝细胞损伤模型组中差异表达的circRNA共10248个,其中表达上调的circRNA共2320个,表达下调的circRNA共7928个。ADLI患者血浆中差异表达的circRNA共6661个,其中表达上调的circRNA共273个,表达下调的circRNA共6388个。在两药和三药联合肝细胞损伤模型以及ADLI患者血浆中均差异表达的circRNA共112个;其中在两药组和ADLI患者血浆中均上调倍数最大的是hsa_circ_0010996,在三药组中上调倍数最大的是hsa_circ_0033188;分别在两药组、三药组和ADLI患者血浆中下调倍数最大hsa_circ_0012054、hsa_circ_0025088和hsa_circ_0021214。差异表达circRNA靶mi RNA预测结果显示,除hsa_circ_0021214外只预测到92个靶miRNA外其余4个circRNA均预测到100个靶miRNA。差异表达circRNA验证结果显示,hsa_circ_0010996在两药组、三药组和ADLI患者血浆中均表达上调,与芯片筛选结果一致。结论在两药联合和三药联合诱导的肝细胞损伤模型中分别筛选到40965个和10248个circRNA,患者血浆中筛选到6661个circRNA。细胞中与血浆中差异表达一致的circRNA有112个。得到验证的circRNA是hsa_circ_0010996,与肝功指标ALT和AST具有相关性,说明其与ADLI密切相关。
[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.
【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】：R52;R575

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