本文選題：尿酸 + 肝細(xì)胞��； 參考：《新疆醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:探討尿酸對肝細(xì)胞氧化應(yīng)激及線粒體功能的影響。方法:體外培養(yǎng)人肝細(xì)胞(L-02),分別加入0、5、10、20、30mg/dL尿酸干預(yù)(0mg/dL為對照組),干預(yù)時間為24、48、72、96h。電鏡觀察肝細(xì)胞形態(tài)及結(jié)構(gòu)。MTT法檢測肝細(xì)胞活力。Annexin V-FITC/PI雙染法,流式檢測肝細(xì)胞凋亡。試劑盒檢測肝細(xì)胞功能指標(biāo)AST和ALT;DNA損傷指標(biāo)8-OhdG;線粒體功能指標(biāo)SDH、CCO和ATP;氧化應(yīng)激指標(biāo)MDA、GSH及SOD的水平。DCFH-DA標(biāo)記,熒光顯微鏡拍照法檢測ROS。Westen blot檢測Nrf2-ARE信號通路下游SOD、GSH和γ-GCS蛋白水平;RT-PCR檢測Nrf2-ARE信號通路下游SOD和GSH的mRNA水平。結(jié)果:1.肝細(xì)胞形態(tài)及凋亡:在尿酸為20mg/dL時肝細(xì)胞出現(xiàn)形狀不規(guī)則、表面絨毛較少,出現(xiàn)凋亡及壞死細(xì)胞;在20mg/dL,作用72~96h后肝細(xì)胞凋亡率大于對照組(P0.05)。2.肝細(xì)胞功能及DNA損傷:ALT、AST在尿酸20mg/dL,作用72~96h后高于對照組(P0.05);8-OhdG隨尿酸水平增加及作用時間延長呈升高趨勢,30mg/dL尿酸組最高;3.肝細(xì)胞氧化應(yīng)激指標(biāo):SOD、GSH在尿酸為20mg/dL,作用48~72h后較對照組降低(P0.05);MDA、ROS在尿酸20mg/dL,作用96h后較對照組均增大(P0.05)。4.肝細(xì)胞Nrf2-ARE下游m RNA水平及蛋白的表達:SOD和GSH mRNA水平在尿酸10mg/dL,作用48~72h后較對照組降低(P0.05);SOD、GSH和γ-GCS蛋白在尿酸為30mg/dL,作用24、72h后較對照組降低(P0.05);5.線粒體功能指標(biāo):ATP、SDH和CCO在尿酸10mg/dL,作用24~72h后高于對照組(P0.05)。結(jié)論:高水平尿酸(20mg/dL)可誘導(dǎo)肝細(xì)胞形態(tài)改變,肝細(xì)胞活力及凋亡增加,并可能通過誘導(dǎo)氧化應(yīng)激加劇對肝細(xì)胞及線粒體功能的損傷。
[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 緯 -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 緯 -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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R575

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