自噬在非酒精性脂肪肝中的作用研究

发布时间：2018-07-15 19:21

【摘要】：研究背景:非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFLD)是指一种排除明确的损肝因素所导致的肝细胞内脂肪积累过度为主要的特性的代谢综合征,与胰岛素抵抗和遗传易感性紧密相关的获得性代谢应激性肝损伤,其中包括单纯性非酒精性脂肪肝(NAFL)、非酒精性脂肪肝炎(NASH)及其相关肝硬化,情况严重者甚至可发展到肝细胞癌。根据流行病学研究分析,在我国目前健康人群体检报告中肝酶学指标异常最常见的病因是是非酒精性脂肪肝,约占总体检人群的90%,而少数的非酒精性脂肪肝炎的患者最终会发展成肝硬化,约占20%。有流行病学数据显示约18%的正常体型和27%的肥胖人群会患有非酒精性脂肪肝病,提示着非酒精性脂肪肝病威胁到人类的健康生存,必须得到我们更多的重视。目前存在多种关于非酒精性脂肪肝发病机理的学说,但具体体制仍不清楚,因此治疗尚停留在减少危险因素如饮食调控,增加运动量减轻体重等,但是缺乏特别有效的药物治疗。因此,我们非常需要对NAFLD的发生、发展及其发病机制进行更深一步的探究。自噬是一种真核细胞通过溶酶体降解受损的细胞器以及清除废弃的蛋白质等功能,在细胞处于应激状态时,可以提供能量和维持稳态。因此,自噬在维持细胞稳态中起到至关重要的作用。自噬的调节在慢性肝病中有发挥着重要的作用。但是,自噬在非酒精性脂肪肝的病程中所发挥的作用仍存在的争议。本课题旨在研究自噬在非酒精性脂肪肝中的作用机制。第一部分非酒精性脂肪肝细胞模型的构建和鉴定目的:构建Huh7细胞及LO2细胞的单纯性非酒精性脂肪变性模型,并进行鉴定模型是否成立。方法:1.使用含10%胎牛血清的DMEM培基常规培养人正常肝细胞LO2细胞和人肝癌细胞Huh7细胞,在细胞融合度达70%-80%时进行细胞的消化传代。正常组换用新鲜的含10%胎牛血清的DMEM培养基继续培养,模型组则换用1m M FFA混合物(油酸:棕榈酸=2:1)的完全培养基,根据FFA的浓度分为5组:200μM、400μM、600μM、800μM,继续培养24小时。2.进行尼罗红染色、荧光流式检测细胞内的脂滴沉积情况,并用MTT法检测细胞活力,并用ROS试剂盒检测细胞氧化应激情况。3.予以Western Blot检测自噬相关蛋白Beclin-1、p62、LC3∥表达水平。结果:1.从荧光显微镜中观察到尼罗红染色中模型组细胞内红染的脂滴相对于正常组明显增多,且成FFA浓度梯度式递增,而正常组未见明显脂滴形成。2.荧光流式细胞检测显示模型组细胞内脂滴沉积比正常组提高,并呈浓度梯度增高。4.正常组与模型组在细胞活力方面无显著性差异。5.ROS实验提示正常组与模型组间无明显变化。6.Western Blot提示模型组中微管相关蛋白LC3II/LC3I及自噬相关蛋白Beclin-1成浓度梯度式增高,而p62则递减,提示非酒精性脂肪肝细胞模型与自噬相关。结论:通过使用混合有FFA的完全培养基可诱导细胞构建类似人单纯性脂肪肝病理改变的肝细胞单纯性脂肪变性模型,并且最高浓度的FFA对Huh7细胞和LO2细胞均无细胞毒性。第二部分自噬在非酒精性脂肪肝中的作用目的:观察通过自噬抑制剂3-Methyladenine(3-MA,3-甲基腺嘌呤,自噬抑制剂)的干预对Huh7细胞和LO2细胞的非酒精性脂肪肝细胞模型的脂滴的沉积的影响,从而了解自噬与非酒精性脂肪肝的相关性,并且自噬在非酒精性脂肪肝中的作用。方法:1.取LO2细胞和Huh7细胞常规培养24小时,等到细胞融合度达70%-80%时进行消化传代,接着分为4组:正常组、3-MA(5μM)组、FFA(600μM)组、3-MA(μM)+FFA(600μM)组,并以此予以相应的处理,继续培养24小时。2.取上述处理后的4组细胞,予以Western Blot检测微管蛋白LC3II蛋白表达水平,明确3-MA抑制剂的处理效果。3.取上述处理后的4组细胞,分别进行尼罗红染色、荧光细胞流式检测,观察细胞内脂滴积累程度的变化,并用甘油三酯试剂盒检测细胞内甘油三酯水平。结果:1.Western Blot检测提示,通过加入3-MA干预后,微管蛋白LC3II明显下降,提示3-MA自噬抑制剂抑制效果明显。2.尼罗红染色及荧光细胞流式检测提示,相比FFA组,同时加入FFA及3-MA,LO2细胞和Huh7细胞内脂滴数量明显减少。3-MA组跟正常组相比无明显变化。细胞内甘油三脂测定的结果与尼罗红染色结果基本一致(p0.05)。结论:通过3-MA自噬抑制剂的干预,非酒精性脂肪肝细胞模型细胞内的脂滴数量有明显减少,并由此可推断出自噬可能有促进NAFLD细胞模型中脂质沉积的作用。全文结论1.利用混有FFA的完全培养基(OA:PA=2:1)培养肝细胞24小时,能够构造出类似人非酒精性脂肪肝病理改变的细胞模型;2.3-MA自噬抑制剂能够减轻非酒精性脂肪肝中细胞的脂质沉积,提示自噬可促进单纯性脂肪肝的病情发展。3.自噬在将来有可能成为治疗非酒精性脂肪肝的新靶点。
[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.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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