自噬在非酒精性脂肪肝中的作用研究
發(fā)布時(shí)間：2018-07-15 19:21
【摘要】：研究背景:非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFLD)是指一種排除明確的損肝因素所導(dǎo)致的肝細(xì)胞內(nèi)脂肪積累過度為主要的特性的代謝綜合征,與胰島素抵抗和遺傳易感性緊密相關(guān)的獲得性代謝應(yīng)激性肝損傷,其中包括單純性非酒精性脂肪肝(NAFL)、非酒精性脂肪肝炎(NASH)及其相關(guān)肝硬化,情況嚴(yán)重者甚至可發(fā)展到肝細(xì)胞癌。根據(jù)流行病學(xué)研究分析,在我國(guó)目前健康人群體檢報(bào)告中肝酶學(xué)指標(biāo)異常最常見的病因是是非酒精性脂肪肝,約占總體檢人群的90%,而少數(shù)的非酒精性脂肪肝炎的患者最終會(huì)發(fā)展成肝硬化,約占20%。有流行病學(xué)數(shù)據(jù)顯示約18%的正常體型和27%的肥胖人群會(huì)患有非酒精性脂肪肝病,提示著非酒精性脂肪肝病威脅到人類的健康生存,必須得到我們更多的重視。目前存在多種關(guān)于非酒精性脂肪肝發(fā)病機(jī)理的學(xué)說,但具體體制仍不清楚,因此治療尚停留在減少危險(xiǎn)因素如飲食調(diào)控,增加運(yùn)動(dòng)量減輕體重等,但是缺乏特別有效的藥物治療。因此,我們非常需要對(duì)NAFLD的發(fā)生、發(fā)展及其發(fā)病機(jī)制進(jìn)行更深一步的探究。自噬是一種真核細(xì)胞通過溶酶體降解受損的細(xì)胞器以及清除廢棄的蛋白質(zhì)等功能,在細(xì)胞處于應(yīng)激狀態(tài)時(shí),可以提供能量和維持穩(wěn)態(tài)。因此,自噬在維持細(xì)胞穩(wěn)態(tài)中起到至關(guān)重要的作用。自噬的調(diào)節(jié)在慢性肝病中有發(fā)揮著重要的作用。但是,自噬在非酒精性脂肪肝的病程中所發(fā)揮的作用仍存在的爭(zhēng)議。本課題旨在研究自噬在非酒精性脂肪肝中的作用機(jī)制。第一部分非酒精性脂肪肝細(xì)胞模型的構(gòu)建和鑒定目的:構(gòu)建Huh7細(xì)胞及LO2細(xì)胞的單純性非酒精性脂肪變性模型,并進(jìn)行鑒定模型是否成立。方法:1.使用含10%胎牛血清的DMEM培基常規(guī)培養(yǎng)人正常肝細(xì)胞LO2細(xì)胞和人肝癌細(xì)胞Huh7細(xì)胞,在細(xì)胞融合度達(dá)70%-80%時(shí)進(jìn)行細(xì)胞的消化傳代。正常組換用新鮮的含10%胎牛血清的DMEM培養(yǎng)基繼續(xù)培養(yǎng),模型組則換用1m M FFA混合物(油酸:棕櫚酸=2:1)的完全培養(yǎng)基,根據(jù)FFA的濃度分為5組:200μM、400μM、600μM、800μM,繼續(xù)培養(yǎng)24小時(shí)。2.進(jìn)行尼羅紅染色、熒光流式檢測(cè)細(xì)胞內(nèi)的脂滴沉積情況,并用MTT法檢測(cè)細(xì)胞活力,并用ROS試劑盒檢測(cè)細(xì)胞氧化應(yīng)激情況。3.予以Western Blot檢測(cè)自噬相關(guān)蛋白Beclin-1、p62、LC3∥表達(dá)水平。結(jié)果:1.從熒光顯微鏡中觀察到尼羅紅染色中模型組細(xì)胞內(nèi)紅染的脂滴相對(duì)于正常組明顯增多,且成FFA濃度梯度式遞增,而正常組未見明顯脂滴形成。2.熒光流式細(xì)胞檢測(cè)顯示模型組細(xì)胞內(nèi)脂滴沉積比正常組提高,并呈濃度梯度增高。4.正常組與模型組在細(xì)胞活力方面無顯著性差異。5.ROS實(shí)驗(yàn)提示正常組與模型組間無明顯變化。6.Western Blot提示模型組中微管相關(guān)蛋白LC3II/LC3I及自噬相關(guān)蛋白Beclin-1成濃度梯度式增高,而p62則遞減,提示非酒精性脂肪肝細(xì)胞模型與自噬相關(guān)。結(jié)論:通過使用混合有FFA的完全培養(yǎng)基可誘導(dǎo)細(xì)胞構(gòu)建類似人單純性脂肪肝病理改變的肝細(xì)胞單純性脂肪變性模型,并且最高濃度的FFA對(duì)Huh7細(xì)胞和LO2細(xì)胞均無細(xì)胞毒性。第二部分自噬在非酒精性脂肪肝中的作用目的:觀察通過自噬抑制劑3-Methyladenine(3-MA,3-甲基腺嘌呤,自噬抑制劑)的干預(yù)對(duì)Huh7細(xì)胞和LO2細(xì)胞的非酒精性脂肪肝細(xì)胞模型的脂滴的沉積的影響,從而了解自噬與非酒精性脂肪肝的相關(guān)性,并且自噬在非酒精性脂肪肝中的作用。方法:1.取LO2細(xì)胞和Huh7細(xì)胞常規(guī)培養(yǎng)24小時(shí),等到細(xì)胞融合度達(dá)70%-80%時(shí)進(jìn)行消化傳代,接著分為4組:正常組、3-MA(5μM)組、FFA(600μM)組、3-MA(μM)+FFA(600μM)組,并以此予以相應(yīng)的處理,繼續(xù)培養(yǎng)24小時(shí)。2.取上述處理后的4組細(xì)胞,予以Western Blot檢測(cè)微管蛋白LC3II蛋白表達(dá)水平,明確3-MA抑制劑的處理效果。3.取上述處理后的4組細(xì)胞,分別進(jìn)行尼羅紅染色、熒光細(xì)胞流式檢測(cè),觀察細(xì)胞內(nèi)脂滴積累程度的變化,并用甘油三酯試劑盒檢測(cè)細(xì)胞內(nèi)甘油三酯水平。結(jié)果:1.Western Blot檢測(cè)提示,通過加入3-MA干預(yù)后,微管蛋白LC3II明顯下降,提示3-MA自噬抑制劑抑制效果明顯。2.尼羅紅染色及熒光細(xì)胞流式檢測(cè)提示,相比FFA組,同時(shí)加入FFA及3-MA,LO2細(xì)胞和Huh7細(xì)胞內(nèi)脂滴數(shù)量明顯減少。3-MA組跟正常組相比無明顯變化。細(xì)胞內(nèi)甘油三脂測(cè)定的結(jié)果與尼羅紅染色結(jié)果基本一致(p0.05)。結(jié)論:通過3-MA自噬抑制劑的干預(yù),非酒精性脂肪肝細(xì)胞模型細(xì)胞內(nèi)的脂滴數(shù)量有明顯減少,并由此可推斷出自噬可能有促進(jìn)NAFLD細(xì)胞模型中脂質(zhì)沉積的作用。全文結(jié)論1.利用混有FFA的完全培養(yǎng)基(OA:PA=2:1)培養(yǎng)肝細(xì)胞24小時(shí),能夠構(gòu)造出類似人非酒精性脂肪肝病理改變的細(xì)胞模型;2.3-MA自噬抑制劑能夠減輕非酒精性脂肪肝中細(xì)胞的脂質(zhì)沉積,提示自噬可促進(jìn)單純性脂肪肝的病情發(fā)展。3.自噬在將來有可能成為治療非酒精性脂肪肝的新靶點(diǎn)。
[Abstract]:Background: nonalcoholic fatty liver disease (NAFLD) refers to a metabolic syndrome that excludes clear liver damage caused by the excessive accumulation of fat in the liver cells, which is closely related to insulin resistance and genetic susceptibility, including simple metabolic stress liver injury, which includes simplex Sexual non-alcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH) and related liver cirrhosis can even develop to hepatocellular carcinoma. According to the epidemiological study, the most common cause of abnormal liver enzyme index in health examination reports in our country is non-alcoholic fatty liver, which accounts for about 90% of the total physical examination population. And a few patients with nonalcoholic steatohepatitis will eventually develop into cirrhosis, accounting for about 20%. epidemiological data showing about 18% of the normal body shape and 27% of the obese people with nonalcoholic fatty liver disease, suggesting that nonalcoholic fatty liver disease threatens the healthy survival of human beings. We must pay more attention to it. The theory of the pathogenesis of nonalcoholic fatty liver disease is still not clear, so the treatment remains to reduce risk factors such as diet regulation, increase exercise and weight loss, but lack of special effective drug treatment. Therefore, we need to take a deeper step on the occurrence, development and pathogenesis of NAFLD. Autophagy is an important function of autophagy in the maintenance of cell homeostasis. Autophagy plays an important role in chronic liver disease. However, the role of autophagy in the course of nonalcoholic fatty liver disease is still in dispute. The purpose of this study is to study the mechanism of autophagy in nonalcoholic fatty liver. Part 1 construction and identification of nonalcoholic fatty liver cell model: Construction of simple nonalcoholic steatosis in Huh7 and LO2 cells The model was established. Methods: 1. the normal human hepatocyte LO2 cells and human hepatocarcinoma cell Huh7 cells were cultured by DMEM with 10% fetal bovine serum, and the cells were digested and passed on the cells when the cell fusion degree was up to 70%-80%. The normal group was replaced by the fresh DMEM medium containing 10% fetal bovine serum, and the model group was the same as the model group. The complete medium of 1m M FFA mixture (oleic acid: palmitic acid =2:1) was divided into 5 groups according to the concentration of FFA: 200 M, 400 mu M, 600 mu M, 800 micron, and continued to culture 24 hour.2. for Nile red staining, fluorescence flow detection of lipid droplet deposition in cells, and MTT method to detect cell viability and detection of cell oxidative stress with ROS Kit Western Blot was used to detect the expression level of autophagy related protein Beclin-1, p62 and LC3. Results: 1. from the fluorescence microscope, the red stained lipid droplets in the cells of the Nile red staining were significantly increased compared to the normal group, and the FFA concentration gradient increased, while the normal group did not have obvious lipid droplets to form the.2. fluorescence flow cytometry display module. There was no significant difference in cell viability between the normal group and the model group with the increase of lipid droplet deposition in the cells of the type group, and there was no significant difference between the model group and the normal group..5.ROS experiment suggested that there was no obvious change between the normal group and the model group..6.Western Blot suggested that the microtubule related protein LC3II/LC3I and the autophagy associated protein Beclin-1 in the model group were in a gradient of concentration in the model group. Higher, and p62 decreased, suggesting that the nonalcoholic fatty liver cell model was associated with autophagy. Conclusion: the complete culture base of mixed FFA can induce cells to construct a simple fatty degeneration model of hepatocytes similar to human simple fatty liver pathology, and the highest concentration of FFA has no cytotoxicity to Huh7 and LO2 cells. Two part of the role of autophagy in nonalcoholic fatty liver purpose: To observe the effect of the intervention of autophagy inhibitor 3-Methyladenine (3-MA, 3- methyl adenine, autophagic inhibitor) on the deposition of lipid droplets in non-alcoholic fatty liver cell models of Huh7 cells and LO2 cells, and to understand the correlation between autophagy and non-alcoholic fatty liver disease. The role of autophagy in nonalcoholic fatty liver. Methods: 1. take LO2 cells and Huh7 cells for 24 hours, and wait until the fusion degree of 70%-80% to be digested, then divide into 4 groups: normal group, 3-MA (5 mu M) group, FFA (600 mu M) group, 3-MA (Muu) +FFA (600 mu M) group, and take the corresponding treatment for 24 hours to take the above.2. to take the above After the 4 groups of cells, the expression level of microtubulin LC3II protein was detected by Western Blot, and the treatment effect of 3-MA inhibitor was determined by using Nile red staining and fluorescence cell flow detection to observe the changes of lipid droplet accumulation in cells, and the triglyceride kit was used to detect triglycerides in cells. Results: results: 1.Western Blot detection suggested that microtubulin LC3II decreased obviously by adding 3-MA, suggesting that the inhibition effect of 3-MA autophagy inhibitor was obviously.2. Nile red staining and fluorescence cell flow cytometry, compared with FFA group, the number of FFA and 3-MA, LO2 cells and Huh7 cells decreased significantly in.3-MA group and normal group. The results of intracellular glycerol three fat determination were consistent with the results of Nile red staining (P0.05). Conclusion: by the intervention of 3-MA autophagy inhibitors, the number of lipid droplets in the cells of non-alcoholic fatty liver cells was significantly reduced, and it could be concluded that autophagy may promote lipid deposition in the NAFLD cell model. Conclusion 1. using the full medium (OA:PA=2:1) mixed with FFA for 24 hours, we can construct a cell model similar to the pathological changes of human nonalcoholic fatty liver. The 2.3-MA autophagy inhibitor can reduce the lipid deposition of cells in nonalcoholic fatty liver, suggesting that autophagy can promote the development of.3. in simple fatty liver. In the future, it may become a new target for the treatment of nonalcoholic fatty liver disease.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575
本文編號(hào)：2125124
[Abstract]:Background: nonalcoholic fatty liver disease (NAFLD) refers to a metabolic syndrome that excludes clear liver damage caused by the excessive accumulation of fat in the liver cells, which is closely related to insulin resistance and genetic susceptibility, including simple metabolic stress liver injury, which includes simplex Sexual non-alcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH) and related liver cirrhosis can even develop to hepatocellular carcinoma. According to the epidemiological study, the most common cause of abnormal liver enzyme index in health examination reports in our country is non-alcoholic fatty liver, which accounts for about 90% of the total physical examination population. And a few patients with nonalcoholic steatohepatitis will eventually develop into cirrhosis, accounting for about 20%. epidemiological data showing about 18% of the normal body shape and 27% of the obese people with nonalcoholic fatty liver disease, suggesting that nonalcoholic fatty liver disease threatens the healthy survival of human beings. We must pay more attention to it. The theory of the pathogenesis of nonalcoholic fatty liver disease is still not clear, so the treatment remains to reduce risk factors such as diet regulation, increase exercise and weight loss, but lack of special effective drug treatment. Therefore, we need to take a deeper step on the occurrence, development and pathogenesis of NAFLD. Autophagy is an important function of autophagy in the maintenance of cell homeostasis. Autophagy plays an important role in chronic liver disease. However, the role of autophagy in the course of nonalcoholic fatty liver disease is still in dispute. The purpose of this study is to study the mechanism of autophagy in nonalcoholic fatty liver. Part 1 construction and identification of nonalcoholic fatty liver cell model: Construction of simple nonalcoholic steatosis in Huh7 and LO2 cells The model was established. Methods: 1. the normal human hepatocyte LO2 cells and human hepatocarcinoma cell Huh7 cells were cultured by DMEM with 10% fetal bovine serum, and the cells were digested and passed on the cells when the cell fusion degree was up to 70%-80%. The normal group was replaced by the fresh DMEM medium containing 10% fetal bovine serum, and the model group was the same as the model group. The complete medium of 1m M FFA mixture (oleic acid: palmitic acid =2:1) was divided into 5 groups according to the concentration of FFA: 200 M, 400 mu M, 600 mu M, 800 micron, and continued to culture 24 hour.2. for Nile red staining, fluorescence flow detection of lipid droplet deposition in cells, and MTT method to detect cell viability and detection of cell oxidative stress with ROS Kit Western Blot was used to detect the expression level of autophagy related protein Beclin-1, p62 and LC3. Results: 1. from the fluorescence microscope, the red stained lipid droplets in the cells of the Nile red staining were significantly increased compared to the normal group, and the FFA concentration gradient increased, while the normal group did not have obvious lipid droplets to form the.2. fluorescence flow cytometry display module. There was no significant difference in cell viability between the normal group and the model group with the increase of lipid droplet deposition in the cells of the type group, and there was no significant difference between the model group and the normal group..5.ROS experiment suggested that there was no obvious change between the normal group and the model group..6.Western Blot suggested that the microtubule related protein LC3II/LC3I and the autophagy associated protein Beclin-1 in the model group were in a gradient of concentration in the model group. Higher, and p62 decreased, suggesting that the nonalcoholic fatty liver cell model was associated with autophagy. Conclusion: the complete culture base of mixed FFA can induce cells to construct a simple fatty degeneration model of hepatocytes similar to human simple fatty liver pathology, and the highest concentration of FFA has no cytotoxicity to Huh7 and LO2 cells. Two part of the role of autophagy in nonalcoholic fatty liver purpose: To observe the effect of the intervention of autophagy inhibitor 3-Methyladenine (3-MA, 3- methyl adenine, autophagic inhibitor) on the deposition of lipid droplets in non-alcoholic fatty liver cell models of Huh7 cells and LO2 cells, and to understand the correlation between autophagy and non-alcoholic fatty liver disease. The role of autophagy in nonalcoholic fatty liver. Methods: 1. take LO2 cells and Huh7 cells for 24 hours, and wait until the fusion degree of 70%-80% to be digested, then divide into 4 groups: normal group, 3-MA (5 mu M) group, FFA (600 mu M) group, 3-MA (Muu) +FFA (600 mu M) group, and take the corresponding treatment for 24 hours to take the above.2. to take the above After the 4 groups of cells, the expression level of microtubulin LC3II protein was detected by Western Blot, and the treatment effect of 3-MA inhibitor was determined by using Nile red staining and fluorescence cell flow detection to observe the changes of lipid droplet accumulation in cells, and the triglyceride kit was used to detect triglycerides in cells. Results: results: 1.Western Blot detection suggested that microtubulin LC3II decreased obviously by adding 3-MA, suggesting that the inhibition effect of 3-MA autophagy inhibitor was obviously.2. Nile red staining and fluorescence cell flow cytometry, compared with FFA group, the number of FFA and 3-MA, LO2 cells and Huh7 cells decreased significantly in.3-MA group and normal group. The results of intracellular glycerol three fat determination were consistent with the results of Nile red staining (P0.05). Conclusion: by the intervention of 3-MA autophagy inhibitors, the number of lipid droplets in the cells of non-alcoholic fatty liver cells was significantly reduced, and it could be concluded that autophagy may promote lipid deposition in the NAFLD cell model. Conclusion 1. using the full medium (OA:PA=2:1) mixed with FFA for 24 hours, we can construct a cell model similar to the pathological changes of human nonalcoholic fatty liver. The 2.3-MA autophagy inhibitor can reduce the lipid deposition of cells in nonalcoholic fatty liver, suggesting that autophagy can promote the development of.3. in simple fatty liver. In the future, it may become a new target for the treatment of nonalcoholic fatty liver disease.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575
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