本文關(guān)鍵詞：利福平誘導(dǎo)小鼠脂肪肝及部分作用機(jī)理　出處：《安徽醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

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【摘要】：背景和目的利福平是臨床廣泛應(yīng)用的抗結(jié)核藥物,眾所周知利福平具有肝毒性。以往臨床關(guān)注的是利福平對(duì)肝細(xì)胞的損傷,而利福平對(duì)肝臟脂質(zhì)代謝的影響及其機(jī)制鮮有報(bào)道。本課題擬在建立利福平誘導(dǎo)小鼠脂肪肝實(shí)驗(yàn)?zāi)Ｐ偷幕A(chǔ)上,觀察利福平引起的小鼠肝臟脂肪變及其動(dòng)態(tài)演變過程,并進(jìn)一步探討其可能的發(fā)生機(jī)制。方法為觀察利福平引起的小鼠肝脂肪變,將42只健康雄性成年小鼠隨機(jī)分成7組,分別為對(duì)照組,利福平處理8小時(shí)(8 h)組、24小時(shí)(24 h)組、3天(3 d)組、1周(1 wk)組、2周(2 wk)組、4周(4 wk)組,其中8 h組、24 h組于清晨9點(diǎn)鐘灌胃給予利福平200 mg/kg一次后于對(duì)應(yīng)時(shí)點(diǎn)剖殺取材;3 d組、1 wk組、2 wk組、4 wk組均于每日清晨9點(diǎn)鐘連續(xù)經(jīng)口灌胃給予利福平200 mg/kg,后于相應(yīng)時(shí)點(diǎn)剖殺取材。所有小鼠剖殺前均禁食6小時(shí)。采集小鼠血及肝臟組織,稱取小鼠肝臟重量。計(jì)算小鼠肝臟系數(shù);分離血清檢測(cè)小鼠血清丙氨酸氨基轉(zhuǎn)移酶(ALT)的水平;HE染色法觀察小鼠肝臟組織病理學(xué)改變;檢測(cè)小鼠血清中膽固醇酯(TCH),甘油三酯(TG),低密度脂蛋白(VLDL),高密度脂蛋白(HDL)水平;檢測(cè)肝臟組織中TG相對(duì)含量及總含量;用油紅O染色法檢測(cè)小鼠肝臟組織中肝臟脂質(zhì)沉積情況。選取對(duì)照組、利福平處理3 d組、1 wk組、4 wk組,用RT-q PCR方法檢測(cè)肝細(xì)胞中參與肝臟脂質(zhì)從頭合成的酶及調(diào)節(jié)因子(Acc、Fas及Scd-1),脂肪酸氧化相關(guān)酶(Cpt-1α、Cyp4a10和Cyp4a14),脂肪酸攝取相關(guān)酶及調(diào)節(jié)因子(Cd36、Fatp、L-fabp)和Pparγm RNA水平;用蛋白質(zhì)免疫印跡技術(shù)檢測(cè)小鼠肝細(xì)胞中SREBP-1c、LXR-α、PXR、PPARγ核蛋白及PPARγ總蛋白表達(dá)水平。結(jié)果與對(duì)照組相比,利福平處理各組小鼠肝臟重量、肝臟系數(shù)從24 h組開始均呈逐漸升高趨勢(shì),于4 wk組達(dá)峰值。利福平處理24 h,3 d及1 wk組ALT水平較對(duì)照組升高;而利福平處理4 wk組較其顯著升高。HE染色結(jié)果顯示,利福平處理8 h、24 h組肝臟組織與對(duì)照組未見明顯變化;利福平處理3 d組可見較多小的圓形空泡;而從1 wk組開始,小鼠肝組織可觀察到明顯增多的大的圓形空泡。利福平處理8 h,24 h組小鼠血清TG,VLDL水平較對(duì)照組升高,3 d組時(shí)顯著升高,具有統(tǒng)計(jì)學(xué)意義,而1 wk組開始則較對(duì)照組呈顯著下降趨勢(shì)。與對(duì)照組相比利福平處理各組小鼠血清中TCH,HDL則呈逐漸降低趨勢(shì)。利福平處理小鼠各組織TG含量則從8 h組即升高,1 wk時(shí)最明顯。肝臟組織油紅O染色顯示,利福平處理8 h、24 h組肝組織可見少量圓形脂滴;3 d組開始出現(xiàn)顯著增加的大的圓形脂滴。用RT-q PCR技術(shù)檢測(cè)發(fā)現(xiàn),利福平處理3 d、1 wk、4 wk組與脂肪酸從頭合成有關(guān)的酶Acc、Fas、Scd-1 m RNA表達(dá)水平較對(duì)照組均明顯上調(diào);進(jìn)一步檢測(cè)SREBP-1c核蛋白表達(dá)水平,利福平處理各組SREBP-1c核蛋白表達(dá)水平與對(duì)照組相比無明顯變化,另一上游調(diào)節(jié)因子LXR-α核蛋白表達(dá)水平較對(duì)照組也無顯著變化;用RT-q PCR技術(shù)檢測(cè)各組與脂肪酸氧化相關(guān)酶Cpt-1α、Cyp4a10及Cyp4a14 m RNA表達(dá)水平,利福平處理各組Cpt-1α、Cyp4a10及Cyp4a14 m RNA呈逐漸上調(diào)趨勢(shì);用RT-q PCR技術(shù)檢測(cè)各組與脂肪酸攝取有關(guān)酶Cd36、Fatp及L-Ffabp m RNA表達(dá)水平,利福平處理各組Cd36、Fatp m RNA表達(dá)水平較對(duì)照組是上調(diào)的,而L-Ffabp m RNA表達(dá)水平較對(duì)照組無明顯變化;檢測(cè)肝臟組織中PPARγm RNA、總蛋白及核蛋白表達(dá)水平,其表達(dá)較對(duì)照組均明顯上調(diào);進(jìn)一步檢測(cè)肝臟組織內(nèi)PXR核蛋白水平及其下游目的基因Cyp3a11 m RNA表達(dá)水平,利福平處理各組PXR核蛋白表達(dá)水平均較對(duì)照組明顯增多,Cyp3a11 m RNA表達(dá)水平較對(duì)照組明顯上調(diào)。結(jié)論1.利福平能引起小鼠肝臟組織脂質(zhì)沉積,且具有時(shí)間-效應(yīng)關(guān)系;2.利福平通過非依賴SREBP-1c及LXR-α激活途徑上調(diào)參與肝臟脂肪酸從頭合成途徑相關(guān)酶的表達(dá)進(jìn)而促進(jìn)脂質(zhì)合成;3.在利福平誘導(dǎo)的小鼠肝臟脂質(zhì)沉積過程中,由外周攝取進(jìn)入肝細(xì)胞內(nèi)游離脂肪酸的增多可能部分歸因于利福平誘導(dǎo)的小鼠肝內(nèi)PXR的激活及PPARγ的上調(diào);4.利福平誘導(dǎo)的小鼠肝脂質(zhì)沉積不依賴于脂肪酸氧化的減少。
[Abstract]:Background and objective: rifampin is anti tuberculosis drugs in clinical applications, as everyone knows rifampicin with liver toxicity. Previous clinical attention is rifampicin on liver cell damage, and the effect of rifampicin on hepatic lipid metabolism and its mechanism is rarely reported. This topic is based on the establishment of rifampicin induced experimental fatty liver model in mice, the evolution process of mice hepatic steatosis caused by rifampicin and dynamic observation, and further explore its possible mechanisms. Methods for observation of rifampicin induced hepatic steatosis, 42 healthy adult male mice were randomly divided into 7 groups, including control group, rifampicin treatment 8 hours (8 h) group, 24 hours (24 h) group, 3 days group (3 D), 1 weeks (1 wk) group, 2 weeks group (2 wk), 4 weeks (4 wk) group, 8 h group, 24 h group at 9 o'clock in the morning by intragastric administration of rifampicin 200 mg/kg once in the corresponding time were killed; 3 group D 1, w K group, 2 wk group, 4 wk group at 9 o'clock in the morning daily continuous oral intragastric administration of rifampicin 200 mg/kg, at the same time after killing animals. All mice were killed before the mice were fasted for 6 hours. Collection of blood and liver tissue, weighing the weight of liver in mice. Calculation of liver coefficient; serum alanine amino mouse serum transferase (ALT) level; to observe the histological change of the liver in mice with pathological HE staining; detection of serum cholesterol ester (TCH), triglyceride (TG), low density lipoprotein (VLDL), high density lipoprotein (HDL) levels in liver tissue; detection of TG relative content and total content; oil red O staining of liver lipid deposition in liver tissue of mice was detected. The control group, rifampin treated 3 D group, 1 wk group, 4 wk group, with the participation of de novo synthesis of enzymes and lipid regulating factor of RT-q PCR method in the detection of liver cells (Acc, Fas and Scd-1), fat Fatty acid oxidation enzymes (Cpt-1 alpha, Cyp4a10 and Cyp4a14), fatty acid uptake related enzymes and regulatory factors (Cd36, Fatp, L-fabp) and Ppar m RNA gamma level; SREBP-1c detection of mouse liver cell protein immunoblotting technique in LXR- alpha, PXR, PPAR protein and PPAR expression of gamma gamma nuclear total egg white level. Results compared with the control group, rifampicin treatment of mice liver weight, liver coefficient from 24 h group was significantly increased in wk group, 4 reached the peak. Rifampicin treatment for 24 h, 3 D and 1 wk group ALT level was higher than the control group; and 4 wk group compared with the treatment of rifampicin significantly increased.HE the staining results showed that rifampicin treatment for 8 h, and the control group had no obvious change in liver tissue of 24 h group; 3 rifampin treated group D showed many small round vacuoles; and from the 1 wk group, liver tissue of mice can be observed in large round vacuoles increased significantly. Rifampin treatment for 8 h, 24 h mice blood The TG and VLDL levels increased compared with the control group, 3 D group increased significantly, with statistical significance, while the 1 wk group compared with the control group decreased significantly compared with the control group. The serum TCH rifampin treated mice, HDL was gradually decreased. The content of TG fabric rifampin treated mice from 8 the H group is increased 1 wk. The most obvious liver oil red O staining showed that the rifampin treated 8 h liver tissue 24 h group showed a small circular lipid droplets; 3 D group began to appear round large lipid droplets increased significantly. By detection of RT-q PCR technology, rifampicin treatment for 3 D, 1 wk. 4 wk group and fatty acid biosynthesis related enzymes Acc, Fas, Scd-1 m expression level of RNA than in the control group were significantly increased; further detection of SREBP-1c nuclear protein expression level, there was no significant change in the treatment groups of rifampicin SREBP-1c nuclear protein expression levels compared with the control group, another upstream regulator of L Expression of XR- protein levels compared with the control group had no significant change; using RT-q PCR technology to detect fatty acid oxidation related enzymes and Cpt-1 alpha, Cyp4a10 and Cyp4a14 m expression levels of RNA were rifampin treated Cpt-1 alpha, Cyp4a10 Cyp4a14 and m RNA was gradually up-regulated; using RT-q PCR technique to detect fatty acid uptake and about Cd36 Fatp and L-Ffabp m enzyme, the expression level of RNA, rifampin treated Cd36 were Fatp m RNA expression levels than the control group was up-regulated, and the expression level of RNA L-Ffabp m compared with the control group, no significant changes in liver tissue; detection of PPAR gamma m RNA, the expression level of total protein and nuclear protein, its expression compared to the control group to investigate the liver; further detection of PXR nuclear protein levels and its downstream gene Cyp3a11 m expression levels of RNA were rifampin treated PXR nuclear protein expression levels were increased significantly compared with the control group, the expression of Cyp3a11 m RNA The level increased significantly compared with the control group. Conclusion 1. rifampicin can cause hepatic lipid deposition, and has a time effect relationship; 2. rifampicin expression through non dependent SREBP-1c and LXR- alpha activation pathway is involved in the upregulation of hepatic fatty acid biosynthesis pathway enzymes and promote lipid synthesis; 3. in mouse liver lipid deposition induced by rifampicin in the peripheral uptake into hepatocytes in free fatty acids increased and may activate PPAR gamma in mouse liver partly induced by rifampicin PXR increases; reduce the liver lipid deposition induced by rifampicin 4. does not depend on the fatty acid oxidation.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R575.5

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本文編號(hào)：1368691