Caveolin-1在非酒精性脂肪性肝病发病机制中的作用研究
发布时间：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确肝损因素所致的以肝细胞脂肪变性为主要特征的临床病理综合征。随着生活水平的提高和膳食结构的改变,NAFLD患病率近年来逐年增高,我国已达到15%左右,西方发达国家更是高达20%-30%,成为最常见的慢性肝病之一,构成日益严重的社会健康问题。NAFLD包括一系列相互联系的病理改变,从单纯性脂肪肝到脂肪性肝炎(nonalcoholic steatohepatitis, NASH)、肝纤维化和肝硬化。总的来说,单纯性NAFLD是一种良性疾病,而NASH却可以逐渐向肝硬化、肝癌等终末期肝病进展。 NAFLD发生发展机制目前尚未完全明确,其治疗也缺乏特别有效方法。“二次打击,,学说认为,胰岛素抵抗引起的外周脂肪分解增加和高胰岛素血症,是导致肝细胞脂肪变性的首要因素；国外针对这一机制设计的临床试验结果提示改善胰岛素抵抗并非对所有NAFLD患者均有效,其主要原因是“二次打击”学说并不能完全解释NAFLD的全部机制,提示还存在其他机制参与NAFLD的发生发展。NAFLD的发生是肝细胞不断贮积脂质的过程,从肝细胞脂质贮积的分子机制切入或许可为认识NAFLD发生发展机制提供新的线索。微囊蛋白1(Caveolin-1)是分子量为21-24kD的多功能信号蛋白,为小凹家族中最重要的成员,富集于细胞膜特异结构小凹、内质网和高尔基体等,并可在胞浆和胞膜之间穿梭。Caveolin-1的氨基酸序列中包括一段长达33个氨基酸(102-134氨基酸残基)的疏水区域,该疏水区域两端均带有一个脯氨酸残基,借助这两个脯氨酸残基形成一个N末端、C末端均面向胞浆的发夹结构。其中N末端(82-101氨基酸残基)是人类Caveolin-1蛋白序列中高度保守的骨架区域。已有研究显示,Caveolin-1在保持质膜微囊的完整性、小胞运输、信号的传导中均起重要作用[7]。 近年来,Caveolin-1在脂质代谢中的作用受到越来越多的关注。Frank等观察到Caveolin-1表达缺失小鼠高脂饮食喂养后不出现肥胖,提示Caveolin-1是高脂饮食诱发肥胖的关键基因之一。后续动物实验发现,Caveolin-1表达缺失可预防动脉粥样硬化、2型糖尿病等糖脂代谢紊乱疾病的发生,提示Caveolin-1在这些代谢性疾病中的重要作用。 为此,本研究利用高脂饮食建立NAFLD小鼠模型、FFA诱导L02细胞脂肪变性,应用RT-PCR及Western blot方法,检测微囊蛋白-1mRNA及蛋白表达水平,及其对脂质合成相关的固醇调节元件结合蛋白(SREBP-1)、脂肪酸合成酶(FASN)、乙酰辅酶A羧化酶(ACC)、环氧合酶2(COX-2)等的作用,探索Caveolin-1在NAFLD发病中的作用及可能机制。 目的 本研究旨在通过观察NAFLD小鼠肝组织Caveolin-1的mRNA和蛋白的表达情况及FFA诱导脂肪变性的L02细胞中,通过抑制或高表达Caveolin-1观察SREBP-1、FASN、ACC、COX-2的变化及TG浓度变化,探究其对脂质合成的作用。 方法 1在体小鼠模型建立：高脂饮食喂养C57小鼠建立NAFLD动物模型,具体方法为：雄性SPF级C57小鼠根据体重分层,随机化分组,对照组喂养普通饲料,实验组喂养高脂饲料(80.5%普通饲料、2%胆固醇、7%猪油、10%蛋黄粉和0.5%胆盐)。造模第4周,第8周、12周末处死小鼠。 2细胞模型建立：采用软脂酸诱导正常人肝细胞株L02细胞建立NAFLD体外模型,具体方案为：正常人肝细胞株L02细胞用含胎牛血清、青链霉素的MEM培养基传代培养,直至细胞达到80-100%融合度。软脂酸和油酸混合物终浓度1mM(软脂酸：油酸=1:2),添加胎牛血清白蛋白1%加入DMEM培养基中。培养48h后收获。油红O染色观察细胞内脂滴形成情况,收集细胞冻溶液测定其中TG的含量,综合评估建模情况。 3从小鼠肝脏组织中、FFA诱导L02细胞中提取RNA,然后进行逆转录,根据合成的Caveolin-1引物进行实时荧光定量PCR(SYBR法),对Caveolin-1的mRNA在两组小鼠中及FFA诱导L02细胞中的变化进行相对定量分析。 4从小鼠肝脏组织、FFA诱导L02细胞中提取总蛋白,行Western Blot.观察实验组和对照组小鼠肝脏及FFA诱导L02细胞中Caveolin-1在蛋白水平的变化,进一步证实其变化是否与mRNA水平变化相对应。 5运用siRNA和重组腺病毒载体的方法,抑制或增强NAFLD细胞模型Caveolin-1的表达,检测肝细胞的脂肪变性程度,并进一步运用实时荧光定量PCR和Western Blot等方法,检测Caveolin-1表达抑制或增强后,NAFLD细胞模型中SREBP-1、FASN、ACC、COX-2指标的变化及可能机制。 结果 1NAFLD小鼠肝组织Caveolin-1mRNA及蛋白表达比对照组明显下降。 2FFA诱导L02细胞脂变过程中Caveolin-1mRNA及蛋白表达比空白对照组明显下降。 3抑制Caveolin-1后L02细胞脂变加剧,与脂质合成相关的蛋白SREBP-1、 FASN、ACC、COX-2蛋白水平升高,高表达Caveolin-1后L02细胞脂变改善,与脂质合成相关的蛋白SREBP-1、FASN、ACC、COX-2蛋白水平降低 结论 Caveolin-1可能在NAFLD发生、发展中发挥一定的抑制机制。可能通过SREBP-1、FASN、ACC、COX-2等发挥作用。
[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 .
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575
本文编号：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 .
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575
【共引文献】
相关博士学位论文 前1条
1 徐磊;非酒精性脂肪性肝病与内分泌代谢紊乱的关系的研究[D];浙江大学;2014年
相关硕士学位论文 前2条
1 潘月;内质网分子伴侣PDIA3在非酒精性脂肪性肝病中的作用及机制研究[D];浙江大学;2014年
2 冉莉;膳食脂肪酸对非酒精性脂肪性肝病发生发展的影响[D];第三军医大学;2014年
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