Caveolin-1在非酒精性脂肪性肝病發(fā)病機(jī)制中的作用研究
發(fā)布時(shí)間：2018-07-16 23:00
【摘要】：研究背景 非酒精性脂肪性肝病(nonalcoholic fatty liver disease, NAFLD)是指除外酒精和其他明確肝損因素所致的以肝細(xì)胞脂肪變性為主要特征的臨床病理綜合征。隨著生活水平的提高和膳食結(jié)構(gòu)的改變,NAFLD患病率近年來逐年增高,我國(guó)已達(dá)到15%左右,西方發(fā)達(dá)國(guó)家更是高達(dá)20%-30%,成為最常見的慢性肝病之一,構(gòu)成日益嚴(yán)重的社會(huì)健康問題。NAFLD包括一系列相互聯(lián)系的病理改變,從單純性脂肪肝到脂肪性肝炎(nonalcoholic steatohepatitis, NASH)、肝纖維化和肝硬化。總的來說,單純性NAFLD是一種良性疾病,而NASH卻可以逐漸向肝硬化、肝癌等終末期肝病進(jìn)展。 NAFLD發(fā)生發(fā)展機(jī)制目前尚未完全明確,其治療也缺乏特別有效方法�！岸未驌�,,學(xué)說認(rèn)為,胰島素抵抗引起的外周脂肪分解增加和高胰島素血癥,是導(dǎo)致肝細(xì)胞脂肪變性的首要因素；國(guó)外針對(duì)這一機(jī)制設(shè)計(jì)的臨床試驗(yàn)結(jié)果提示改善胰島素抵抗并非對(duì)所有NAFLD患者均有效,其主要原因是“二次打擊”學(xué)說并不能完全解釋NAFLD的全部機(jī)制,提示還存在其他機(jī)制參與NAFLD的發(fā)生發(fā)展。NAFLD的發(fā)生是肝細(xì)胞不斷貯積脂質(zhì)的過程,從肝細(xì)胞脂質(zhì)貯積的分子機(jī)制切入或許可為認(rèn)識(shí)NAFLD發(fā)生發(fā)展機(jī)制提供新的線索。微囊蛋白1(Caveolin-1)是分子量為21-24kD的多功能信號(hào)蛋白,為小凹家族中最重要的成員,富集于細(xì)胞膜特異結(jié)構(gòu)小凹、內(nèi)質(zhì)網(wǎng)和高爾基體等,并可在胞漿和胞膜之間穿梭。Caveolin-1的氨基酸序列中包括一段長(zhǎng)達(dá)33個(gè)氨基酸(102-134氨基酸殘基)的疏水區(qū)域,該疏水區(qū)域兩端均帶有一個(gè)脯氨酸殘基,借助這兩個(gè)脯氨酸殘基形成一個(gè)N末端、C末端均面向胞漿的發(fā)夾結(jié)構(gòu)。其中N末端(82-101氨基酸殘基)是人類Caveolin-1蛋白序列中高度保守的骨架區(qū)域。已有研究顯示,Caveolin-1在保持質(zhì)膜微囊的完整性、小胞運(yùn)輸、信號(hào)的傳導(dǎo)中均起重要作用[7]。 近年來,Caveolin-1在脂質(zhì)代謝中的作用受到越來越多的關(guān)注。Frank等觀察到Caveolin-1表達(dá)缺失小鼠高脂飲食喂養(yǎng)后不出現(xiàn)肥胖,提示Caveolin-1是高脂飲食誘發(fā)肥胖的關(guān)鍵基因之一。后續(xù)動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn),Caveolin-1表達(dá)缺失可預(yù)防動(dòng)脈粥樣硬化、2型糖尿病等糖脂代謝紊亂疾病的發(fā)生,提示Caveolin-1在這些代謝性疾病中的重要作用。 為此,本研究利用高脂飲食建立NAFLD小鼠模型、FFA誘導(dǎo)L02細(xì)胞脂肪變性,應(yīng)用RT-PCR及Western blot方法,檢測(cè)微囊蛋白-1mRNA及蛋白表達(dá)水平,及其對(duì)脂質(zhì)合成相關(guān)的固醇調(diào)節(jié)元件結(jié)合蛋白(SREBP-1)、脂肪酸合成酶(FASN)、乙酰輔酶A羧化酶(ACC)、環(huán)氧合酶2(COX-2)等的作用,探索Caveolin-1在NAFLD發(fā)病中的作用及可能機(jī)制。 目的 本研究旨在通過觀察NAFLD小鼠肝組織Caveolin-1的mRNA和蛋白的表達(dá)情況及FFA誘導(dǎo)脂肪變性的L02細(xì)胞中,通過抑制或高表達(dá)Caveolin-1觀察SREBP-1、FASN、ACC、COX-2的變化及TG濃度變化,探究其對(duì)脂質(zhì)合成的作用。 方法 1在體小鼠模型建立：高脂飲食喂養(yǎng)C57小鼠建立NAFLD動(dòng)物模型,具體方法為：雄性SPF級(jí)C57小鼠根據(jù)體重分層,隨機(jī)化分組,對(duì)照組喂養(yǎng)普通飼料,實(shí)驗(yàn)組喂養(yǎng)高脂飼料(80.5%普通飼料、2%膽固醇、7%豬油、10%蛋黃粉和0.5%膽鹽)。造模第4周,第8周、12周末處死小鼠。 2細(xì)胞模型建立：采用軟脂酸誘導(dǎo)正常人肝細(xì)胞株L02細(xì)胞建立NAFLD體外模型,具體方案為：正常人肝細(xì)胞株L02細(xì)胞用含胎牛血清、青鏈霉素的MEM培養(yǎng)基傳代培養(yǎng),直至細(xì)胞達(dá)到80-100%融合度。軟脂酸和油酸混合物終濃度1mM(軟脂酸：油酸=1:2),添加胎牛血清白蛋白1%加入DMEM培養(yǎng)基中。培養(yǎng)48h后收獲。油紅O染色觀察細(xì)胞內(nèi)脂滴形成情況,收集細(xì)胞凍溶液測(cè)定其中TG的含量,綜合評(píng)估建模情況。 3從小鼠肝臟組織中、FFA誘導(dǎo)L02細(xì)胞中提取RNA,然后進(jìn)行逆轉(zhuǎn)錄,根據(jù)合成的Caveolin-1引物進(jìn)行實(shí)時(shí)熒光定量PCR(SYBR法),對(duì)Caveolin-1的mRNA在兩組小鼠中及FFA誘導(dǎo)L02細(xì)胞中的變化進(jìn)行相對(duì)定量分析。 4從小鼠肝臟組織、FFA誘導(dǎo)L02細(xì)胞中提取總蛋白,行Western Blot.觀察實(shí)驗(yàn)組和對(duì)照組小鼠肝臟及FFA誘導(dǎo)L02細(xì)胞中Caveolin-1在蛋白水平的變化,進(jìn)一步證實(shí)其變化是否與mRNA水平變化相對(duì)應(yīng)。 5運(yùn)用siRNA和重組腺病毒載體的方法,抑制或增強(qiáng)NAFLD細(xì)胞模型Caveolin-1的表達(dá),檢測(cè)肝細(xì)胞的脂肪變性程度,并進(jìn)一步運(yùn)用實(shí)時(shí)熒光定量PCR和Western Blot等方法,檢測(cè)Caveolin-1表達(dá)抑制或增強(qiáng)后,NAFLD細(xì)胞模型中SREBP-1、FASN、ACC、COX-2指標(biāo)的變化及可能機(jī)制。 結(jié)果 1NAFLD小鼠肝組織Caveolin-1mRNA及蛋白表達(dá)比對(duì)照組明顯下降。 2FFA誘導(dǎo)L02細(xì)胞脂變過程中Caveolin-1mRNA及蛋白表達(dá)比空白對(duì)照組明顯下降。 3抑制Caveolin-1后L02細(xì)胞脂變加劇,與脂質(zhì)合成相關(guān)的蛋白SREBP-1、 FASN、ACC、COX-2蛋白水平升高,高表達(dá)Caveolin-1后L02細(xì)胞脂變改善,與脂質(zhì)合成相關(guān)的蛋白SREBP-1、FASN、ACC、COX-2蛋白水平降低 結(jié)論 Caveolin-1可能在NAFLD發(fā)生、發(fā)展中發(fā)揮一定的抑制機(jī)制�？赡芡ㄟ^SREBP-1、FASN、ACC、COX-2等發(fā)揮作用。
[Abstract]:Background of the study
Non - alcoholic fatty liver disease refers to the clinical pathological syndrome characterized by fatty degeneration of liver cells caused by alcohol and other specific liver loss factors . With the improvement of living standard and the change of dietary structure , the prevalence of the disease is about 15 % in China and 20 % -30 % in the developed countries .
It is suggested that it is not an effective way to develop the mechanism of fatty degeneration of liver cells . The main reason is that the theory of secondary attack is the most important factor in the development of fatty degeneration of hepatocytes .
In recent years , the role of Caveolin - 1 in lipid metabolism has been paid more and more attention . Frank et al . observed that Caveolin - 1 was one of the key genes in high - fat diet induced obesity , suggesting that Caveolin - 1 was one of the key genes in high - fat diet - induced obesity . It was found that the deletion of Caveolin - 1 could prevent the occurrence of glycolipid metabolism disorders , such as atherosclerosis and type 2 diabetes , suggesting that Caveolin - 1 plays an important role in these metabolic diseases .
In this study , the effects of Caveolin - 1 on the pathogenesis and possible mechanism of Caveolin - 1 were investigated by using high - fat diet to establish the model of LD mice , FFA - induced fatty degeneration of L02 cells , RT - PCR and Western blot to detect the level of protein - 1 mRNA and protein expression , and the role of sterol regulatory element - binding protein , fatty acid synthetase ( FASN ) , acetyl - CoA Carboxylase ( ACC ) and cyclooxygenase - 2 ( COX - 2 ) associated with lipid synthesis .
Purpose
The purpose of this study was to investigate the effects of Caveolin - 1 on lipid synthesis by observing the expression of Caveolin - 1 mRNA and protein and FFA - induced fatty degeneration in L02 cells induced by FFA .
method
1 in vivo mouse model : A high - fat diet was used to feed C57 mice to establish an animal model of naf LD . The specific methods were as follows : male SPF C57 mice were randomly divided into groups according to weight stratification , randomization group and control group . The experimental group fed high fat feed ( 80.5 % ordinary feed , 2 % cholesterol , 7 % lard , 10 % yolk powder and 0.5 % bile salt ) . Mice were sacrificed at Week 4 , Week 8 , and 12 .
2 - cell model was established by using palmitic acid to induce L02 cells of normal human liver cell line to establish an in vitro model of naf LD . The specific protocol was as follows : normal human liver cell line L02 cells were passaged with MEM medium containing fetal bovine serum and penicillin streptomycin until the cells reached 80 - 100 % fusion degree . The final concentration of palmitic acid and oleic acid was 1 mM ( palmitic acid : oleic acid = 1 : 2 ) . After 48 hours of culture , the content of lipid droplets in the cells was observed and the model was evaluated comprehensively .
3 From the mouse liver tissues , the RNA was extracted from FFA - induced L02 cells , then reverse transcription was carried out . According to the synthesized Caveolin - 1 primer , real - time fluorescence quantitative PCR ( SYBR method ) was carried out , and the changes of Caveolin - 1 mRNA in two groups of mice and FFA - induced L02 cells were analyzed quantitatively .
The changes of Caveolin - 1 and Caveolin - 1 in liver and FFA - induced L02 cells in experimental group and control group were observed by Western Blot .
5 . Using siRNA and recombinant adenovirus vector , the expression of Caveolin - 1 was inhibited or enhanced . The degree of fatty degeneration of hepatocytes was detected , and the changes and possible mechanism of the expression of Caveolin - 1 , FASN , ACC and COX - 2 were detected by real - time fluorescence quantitative PCR and Western Blot .
Results
The expression of Caveolin - 1 mRNA and protein in liver tissue was significantly lower than that in the control group .
The expression of Caveolin - 1 mRNA and protein in L02 cells induced by 2FFA was significantly lower than that in blank control group .
3 inhibited the increase of L02 cell lipid after Caveolin - 1 , and the level of the protein related to lipid synthesis increased , the level of FASN , ACC , COX - 2 protein increased , the L02 cell lipid was improved after high expression of Caveolin - 1 , and the level of the protein related to lipid synthesis decreased , and the level of the protein related to lipid synthesis decreased .
Conclusion
Caveolin - 1 may play an important role in the development and development .
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575
本文編號(hào)：2127945
[Abstract]:Background of the study
Non - alcoholic fatty liver disease refers to the clinical pathological syndrome characterized by fatty degeneration of liver cells caused by alcohol and other specific liver loss factors . With the improvement of living standard and the change of dietary structure , the prevalence of the disease is about 15 % in China and 20 % -30 % in the developed countries .
It is suggested that it is not an effective way to develop the mechanism of fatty degeneration of liver cells . The main reason is that the theory of secondary attack is the most important factor in the development of fatty degeneration of hepatocytes .
In recent years , the role of Caveolin - 1 in lipid metabolism has been paid more and more attention . Frank et al . observed that Caveolin - 1 was one of the key genes in high - fat diet induced obesity , suggesting that Caveolin - 1 was one of the key genes in high - fat diet - induced obesity . It was found that the deletion of Caveolin - 1 could prevent the occurrence of glycolipid metabolism disorders , such as atherosclerosis and type 2 diabetes , suggesting that Caveolin - 1 plays an important role in these metabolic diseases .
In this study , the effects of Caveolin - 1 on the pathogenesis and possible mechanism of Caveolin - 1 were investigated by using high - fat diet to establish the model of LD mice , FFA - induced fatty degeneration of L02 cells , RT - PCR and Western blot to detect the level of protein - 1 mRNA and protein expression , and the role of sterol regulatory element - binding protein , fatty acid synthetase ( FASN ) , acetyl - CoA Carboxylase ( ACC ) and cyclooxygenase - 2 ( COX - 2 ) associated with lipid synthesis .
Purpose
The purpose of this study was to investigate the effects of Caveolin - 1 on lipid synthesis by observing the expression of Caveolin - 1 mRNA and protein and FFA - induced fatty degeneration in L02 cells induced by FFA .
method
1 in vivo mouse model : A high - fat diet was used to feed C57 mice to establish an animal model of naf LD . The specific methods were as follows : male SPF C57 mice were randomly divided into groups according to weight stratification , randomization group and control group . The experimental group fed high fat feed ( 80.5 % ordinary feed , 2 % cholesterol , 7 % lard , 10 % yolk powder and 0.5 % bile salt ) . Mice were sacrificed at Week 4 , Week 8 , and 12 .
2 - cell model was established by using palmitic acid to induce L02 cells of normal human liver cell line to establish an in vitro model of naf LD . The specific protocol was as follows : normal human liver cell line L02 cells were passaged with MEM medium containing fetal bovine serum and penicillin streptomycin until the cells reached 80 - 100 % fusion degree . The final concentration of palmitic acid and oleic acid was 1 mM ( palmitic acid : oleic acid = 1 : 2 ) . After 48 hours of culture , the content of lipid droplets in the cells was observed and the model was evaluated comprehensively .
3 From the mouse liver tissues , the RNA was extracted from FFA - induced L02 cells , then reverse transcription was carried out . According to the synthesized Caveolin - 1 primer , real - time fluorescence quantitative PCR ( SYBR method ) was carried out , and the changes of Caveolin - 1 mRNA in two groups of mice and FFA - induced L02 cells were analyzed quantitatively .
The changes of Caveolin - 1 and Caveolin - 1 in liver and FFA - induced L02 cells in experimental group and control group were observed by Western Blot .
5 . Using siRNA and recombinant adenovirus vector , the expression of Caveolin - 1 was inhibited or enhanced . The degree of fatty degeneration of hepatocytes was detected , and the changes and possible mechanism of the expression of Caveolin - 1 , FASN , ACC and COX - 2 were detected by real - time fluorescence quantitative PCR and Western Blot .
Results
The expression of Caveolin - 1 mRNA and protein in liver tissue was significantly lower than that in the control group .
The expression of Caveolin - 1 mRNA and protein in L02 cells induced by 2FFA was significantly lower than that in blank control group .
3 inhibited the increase of L02 cell lipid after Caveolin - 1 , and the level of the protein related to lipid synthesis increased , the level of FASN , ACC , COX - 2 protein increased , the L02 cell lipid was improved after high expression of Caveolin - 1 , and the level of the protein related to lipid synthesis decreased , and the level of the protein related to lipid synthesis decreased .
Conclusion
Caveolin - 1 may play an important role in the development and development .
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575
【共引文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 徐磊;非酒精性脂肪性肝病與內(nèi)分泌代謝紊亂的關(guān)系的研究[D];浙江大學(xué);2014年
相關(guān)碩士學(xué)位論文 前2條
1 潘月;內(nèi)質(zhì)網(wǎng)分子伴侶PDIA3在非酒精性脂肪性肝病中的作用及機(jī)制研究[D];浙江大學(xué);2014年
2 冉莉;膳食脂肪酸對(duì)非酒精性脂肪性肝病發(fā)生發(fā)展的影響[D];第三軍醫(yī)大學(xué);2014年
，本文編號(hào)：2127945
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