尿酸对肝细胞氧化应激及线粒体功能影响的研究

发布时间：2018-06-24 19:27

本文选题：尿酸 + 肝细胞　；参考：《新疆医科大学》2016年硕士论文

【摘要】：目的:探讨尿酸对肝细胞氧化应激及线粒体功能的影响。方法:体外培养人肝细胞(L-02),分别加入0、5、10、20、30mg/dL尿酸干预(0mg/dL为对照组),干预时间为24、48、72、96h。电镜观察肝细胞形态及结构。MTT法检测肝细胞活力。Annexin V-FITC/PI双染法,流式检测肝细胞凋亡。试剂盒检测肝细胞功能指标AST和ALT;DNA损伤指标8-OhdG;线粒体功能指标SDH、CCO和ATP;氧化应激指标MDA、GSH及SOD的水平。DCFH-DA标记,荧光显微镜拍照法检测ROS。Westen blot检测Nrf2-ARE信号通路下游SOD、GSH和γ-GCS蛋白水平;RT-PCR检测Nrf2-ARE信号通路下游SOD和GSH的mRNA水平。结果:1.肝细胞形态及凋亡:在尿酸为20mg/dL时肝细胞出现形状不规则、表面绒毛较少,出现凋亡及坏死细胞;在20mg/dL,作用72~96h后肝细胞凋亡率大于对照组(P0.05)。2.肝细胞功能及DNA损伤:ALT、AST在尿酸20mg/dL,作用72~96h后高于对照组(P0.05);8-OhdG随尿酸水平增加及作用时间延长呈升高趋势,30mg/dL尿酸组最高;3.肝细胞氧化应激指标:SOD、GSH在尿酸为20mg/dL,作用48~72h后较对照组降低(P0.05);MDA、ROS在尿酸20mg/dL,作用96h后较对照组均增大(P0.05)。4.肝细胞Nrf2-ARE下游m RNA水平及蛋白的表达:SOD和GSH mRNA水平在尿酸10mg/dL,作用48~72h后较对照组降低(P0.05);SOD、GSH和γ-GCS蛋白在尿酸为30mg/dL,作用24、72h后较对照组降低(P0.05);5.线粒体功能指标:ATP、SDH和CCO在尿酸10mg/dL,作用24~72h后高于对照组(P0.05)。结论:高水平尿酸(20mg/dL)可诱导肝细胞形态改变,肝细胞活力及凋亡增加,并可能通过诱导氧化应激加剧对肝细胞及线粒体功能的损伤。
[Abstract]:Objective: to investigate the effect of uric acid on oxidative stress and mitochondrial function of hepatocytes. Methods: human hepatocytes (L-02) were cultured in vitro and treated with 30 mg / dL uric acid (0 mg / dL) for 24 minutes and 48 minutes for 7296h, respectively. The morphology and structure of hepatocytes were observed by MTT method. Annexin V-FITC / Pi double staining method was used to detect the viability of hepatocytes, and the apoptosis of hepatocytes was detected by flow cytometry. The liver cell function index AST and alt DNA damage index 8-OhdG, mitochondrial function index SDH CCO and ATP, oxidative stress index MDA-GSH and SOD level. DCFH-DA were detected by Kit. The protein levels of SODH and 纬 -GCS in the downstream of the Nrf2-ARE signaling pathway were detected by fluorescence microscope blot and the mRNA levels of SOD and GSH in the downstream of the Nrf2-ARE signaling pathway were detected by reverse transcription-polymerase chain reaction (RT-PCR). The result is 1: 1. Morphology and apoptosis of hepatocytes: when uric acid was 20 mg / dL, the hepatocytes appeared irregular shape, less villi on the surface, apoptosis and necrotic cells, and at 20 mg / dL, the apoptotic rate of hepatocytes was higher than that of the control group (P0.05). The function of liver cells and DNA damage at 20 mg / dL of uric acid were significantly higher than those of the control group (P0.05) with the increase of uric acid level and the prolongation of the time of action. The highest level was 3mg / dL uric acid group (P < 0.05). The oxidative stress index of liver cells was 20 mg / dL in uric acid, decreased after 48h / 72 h (P0.05), and increased after 96h compared with control group (P0.05). The mRNA level of mRNA and protein in the downstream of Nrf2-are decreased after exposure to uric acid at 10 mg / dL (P0.05), the levels of GSH and 纬 -GCS decreased at the level of 30 mg / dL in uric acid (P0.05), and decreased at 2472 h (P0.05) in comparison with the control group (P0.05). Mitochondrial function index: ATP SDH and CCO were significantly higher than those in control group (P0.05) after exposure to uric acid (10 mg / d L) for 24 h (P < 0.05). Conclusion: high level uric acid (20mg / dL) can induce the morphological changes of hepatocytes, increase the viability and apoptosis of hepatocytes, and aggravate the damage to the function of hepatocytes and mitochondria by inducing oxidative stress.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575

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