MicroRNA与异烟肼所致小鼠抗结核药物性肝损伤关系的研究

发布时间：2018-08-29 19:20

【摘要】：目的采用INH诱导的小鼠抗结核药物性肝损伤模型，观察miR-122、miR-155及氧化损伤指标的动态变化，初步探讨miR-122、miR-155及氧化应激在抗结核药物性肝损伤发生发展中的作用及关系，为抗结核药物性肝损伤的早期诊断和发病机制研究提供实验依据。方法1）急性毒性实验：（1）实验对象：昆明小鼠，对照组（10只）给予生理盐水，实验组（70只）予以INH（180mg/kg）灌胃处理，实验组分别在给药后的0.25、0.75、1.5、6、12、18、24h和对照组24h留取10只小鼠的血清和肝组织标本。（2）实验方法：HE染色法，光学显微镜下观察肝组织病理学变化；全自动生化分析仪检测血清ALT和AST水平；实时荧光定量PCR检测肝组织中miR-122和miR-155表达。（3）统计分析：采用SPSS17.0数据分析，数据以x s表示，计量资料采用单因素方差法，相关性分析采用Pearman相关性分析，P0.05为差异有统计学意义。 2）慢性毒性实验：（1）实验对象：除实验组每天予以INH（90mg/kg）灌胃，其中实验组分别在连续给药后的1D、3D、5D、1W、2W、3W、4W和对照组4W留取小鼠的血清和肝组织标本，其他同急性毒性实验。（2）实验方法：组织病理学、ALT和AST检测同急性毒性实验，，一般生化分析法检测CuZnSOD和MDA的水平；酶联免疫吸附吸附法检测MT和MRPS11蛋白表达水平；miR-122表达水平检测同急性毒性实验。（3）统计分析：同急性毒性实验。结果1）急性毒性实验：（1）肝组织病理学：给药后的0.25h，可见少量肝细胞肿胀，随给药时间的变化并逐渐加重。（2）ALT和AST：小鼠血清中ALT和AST在0.75h出现显著性变化，且两者均上升到最大值。（3）miR-122和miR-155：给药后miR-122整体呈下降趋势，0.25h即开始发生显著性下降为（56.50±27.77）％，（P0.05）；miR-155呈上升趋势，0.75h开始出现显著性上升（11.25±1.43）倍。（4）相关性分析：miR-122/miR-155比值与ALT和AST均呈负相关性（r＝-0.356，r＝-0.246；P0.05），且与肝组织病理学评分有很好的负相关性（r＝-0.779，P0.01）。 2）慢性毒性实验：（1）肝组织病理学：连续给药后的第3D，显微镜下可见少量肝细胞肿胀，胞浆稀疏。随着用药时间延长，肝组织损伤程度逐渐加重。（2）ALT和AST：两者分别在2W和3W出现显著性变化。（3）CuZnSOD和MDA：与对照组相比5D发生了显著性变化。（4）miR-122：给药后整体呈下降趋势，第3D表达开始下降（88.72±5.15）％（P0.05）。（5）MT和MRPS11：与对照组相比两者均在第3D出现变化，且在2W分别下降到最小值（908.68±152.87）pg/ml和（9.72±1.31）pg/ml（P0.01）。（6）相关性分析：miR-122与CuZnSOD、MDA和MT存在相关性（r＝0.240，r＝-0.311，r＝0.415；均P0.05)；MRPS11与CuZnSOD，MDA和MT存在相关性（r＝0.313，r＝-0.250，r＝0.366；均P0.05）。结论急/慢性毒性实验中，miRNA与ADLI存在相关性，miR-122低表达和miR-155高表达均与ADLI发生发展有关，miR-122/miR-155比值有望成为ADLI早期诊断标志物，且miR-122可能通过调节MRPS11参与ADLI中的氧化应激反应，为ADLI发病机制研究提供了新的实验依据。
[Abstract]:Objective to observe the dynamic changes of miR-122,miR-155 and oxidative damage indexes in mice induced by INH, and to explore the role and relationship of miR-122,miR-155 and oxidative stress in the occurrence and development of liver injury induced by antituberculous drugs. To provide experimental evidence for the early diagnosis and pathogenesis of liver injury induced by antituberculous drugs. Methods 1) Acute toxicity test: (1) Experimental subjects: Kunming mice, control group (10 mice) were given normal saline, experimental group (70 mice) were treated by INH (180mg/kg) intragastric perfusion. The samples of serum and liver tissue of 10 mice were collected in the experimental group at 0.250.75 ~ 1.5U 1.5A ~ (12) ~ (12) ~ (18) h after administration for 24 h and in the control group at 24 h. (2) the pathological changes of liver tissue were observed by the method of: (2) the pathological changes of liver tissue were observed under optical microscope, and the levels of ALT and AST in serum were detected by automatic biochemical analyzer. Real-time fluorescence quantitative PCR was used to detect the expression of miR-122 and miR-155 in liver tissue. (3) Statistical analysis: SPSS17.0 data were analyzed, the data were expressed as x s, and the data were measured by single factor variance method. The correlation analysis using Pearman correlation analysis (P0.05) had statistical significance. 2) chronic toxicity experiment: (1) experiment object: except the experimental group was given INH (90mg/kg) daily, The serum and liver tissues of the experimental group were collected after continuous administration of 1DX 3DX 5DX 1W 2WN 3W 4W and the control group 4W respectively. (2) Experimental methods: the detection of alt and AST in histopathology was the same as acute toxicity test. The levels of CuZnSOD and MDA were detected by general biochemical analysis, and the expression levels of MT and MRPS11 protein by Elisa were detected in the same way as acute toxicity test. (3) Statistical analysis: same acute toxicity test. Results 1) Acute toxicity test: (1) liver histopathology: at 0.25 h after administration, a small amount of hepatocyte swelling was observed, which increased gradually with the time of administration. (2) ALT and AST in serum of ALT and AST: mice showed significant changes at 0.75 h. And both of them increased to the maximum. (3) after miR-122 and miR-155: administration, the whole miR-122 showed a downward trend of 0.25 h, which began to significantly decrease to (56.50 卤27.77), (P0.05); (4) the correlation analysis showed that the ratio of 1: miR-122% miR-155 was negatively correlated with ALT and AST (r-0.356r-0.246). (2) chronic toxicity test: (1) liver histopathology: 3D after continuous administration, a small amount of liver cells were swollen and cytoplasm was sparse under microscope. (2) there were significant changes in ALT and AST: at 2W and 3W, respectively. (3) CuZnSOD and MDA: showed significant changes compared with the control group. (4) the whole level of miR-122: decreased. 3D expression began to decrease (88.72 卤5.15)% (P0.05). (5) MT and MRPS11: showed changes in 3D compared with the control group, and decreased to the minimum value (908.68 卤152.87) pg/ml and (9.72 卤1.31) pg/ml (P0.01). (6) at 2W. There was a correlation between MRPS11 and CuZnSOD,MDA and MT (r = 0.313). Conclusion the low expression of miR-122 and the high expression of miR-155 in acute / chronic toxicity test are related to the occurrence and development of ADLI. The ratio of miR-122 / miR-155 may be the early diagnostic marker of ADLI, and miR-122 may participate in the oxidative stress reaction of ADLI by regulating MRPS11. It provides a new experimental basis for the study of the pathogenesis of ADLI.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

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