中碳链脂肪酸改善脂变肝细胞损伤和脂代谢紊乱的机制

发布时间：2018-08-26 11:08

【摘要】：随着人们物质生活水平的提高以及体育活动的减少,导致肥胖及其相关代谢疾病的发病率显著增加,继而损伤机体的健康。所以,近年来寻求一种绿色高效的减肥手段一贯被研究者所重视。然而膳食油脂作为引起体内脂质沉淀的主要原因,对机体的生物学作用和内在分子机制不甚清楚。本论文采用组合生物学技术,对非酒精性脂肪肝病(NAFLD)细胞模型的建立、脂肪酸(FA)对该模型细胞凋亡、氧化压力、免疫调控和脂质代谢的分子作用机制进行了非常全面的探讨。采用MTT细胞活性检测法、LDH细胞坏死检测法、Hoechst 33342染色对细胞凋亡测定法、油红O染色法和甘油三酯(TG)定量测定法、扫描电镜对细胞膜损伤程度检测法对建立符合人类自然病规律的NAFLD模型(最大脂质沉淀和较低的细胞毒性)的条件进行了探索。采用检测细胞凋亡的Annexin V-FITC/PI双染色流式细胞仪法(FCM)和Hoechst33342/PI荧光显微镜法、细胞的抗氧化及氧化压力的酶标仪测定法、差异蛋白测定的蛋白组学法、WB以及ELISA结合荧光定量PCR技术考察了免疫因子的表达,研究膳食中FA的种类对人肝脂变细胞凋亡、免疫调控和氧化压力的影响,探究促进NAFLD到进一步肝损伤的机制。采用MTT细胞活性检测法、油红O染色和TG定性定量测定法以及脂代谢基因型荧光定量PCR、ELISA与WB相结合的方法,对中碳链脂肪酸(MCFA)对NAFLD细胞脂滴的聚集及相关脂代谢的分子作用机制进行了全面的探究。本研究的主要结果总结如下:第1章绪论部分对MCFA的生物学特性和NAFLD的研究进展分别做了综述,并对引起NAFLD的分子作用机制作出了展望。FA生物学特性,包括FA的代谢特点、生理功能、安全性和潜在的局限性的研究进展。NAFLD研究部分包括该病发病的原因、模型的建立、引起的细胞凋亡、氧化压力、免疫调控和相关脂代谢信号通路的研究进展。第2章对建立符合人类自然疾病规律的NAFLD模型的最适条件进行了探索。通过MTT和LDH相互验证的实验方法考察混合FA(油酸:棕榈酸=2:1)和单一FA(油酸)对L02和HepG2两种肝细胞的增殖与凋亡的影响,结果发现FA对细胞的毒性均产生浓度和时间依赖性,当浓度低于400μM时,不会对细胞的活性存在影响,且LO2细胞对FA的毒性影响较HepG2肝细胞更加敏感。进一步通过Hoechst染色法对细胞凋亡的检测结果说明细胞凋亡的程度与FA的浓度呈正相关,在浓度小于400 μM时,与对照组相比均不会明显引起细胞的凋亡;浓度升高到800 μM时,均可引起细胞的毒性。通过油红O染色定性观察细胞内的脂滴及细胞的形态,磷酸甘油氧化酶法定量对胞里TG含量的测定,结果表明FA混合物相对于单一 FA更适合用来建立肝脂变细胞模型;当混合FA与LO2细胞孵育时间为24 h,作用终浓度为200μM 时,到达最高的脂质沉淀且造成的肝细胞慢性损伤和NAFLD的慢性肝脂肪变接近。第3章通过研究膳食中FA的种类对人肝脂变细胞凋亡和氧化压力的影响,探究促进NAFLD到进一步肝损伤的机制。通过对细胞凋亡分析表明:同LCFA对比,MCFA能显著降低早、中、晚期凋亡细胞和坏死细胞的百分比,同时抑制Caspase3和Caspase9酶的活性。氧化分析结果表明:采用蛋白双向电泳及二级质谱方法研究发现LCFA能抑制抗氧化酶的产生;进一步通过对SOD、MDA、GSH和ROS的测定也发现MCFA不会产生氧化压力,而LCFA会对细胞产生氧化压力。本章结果表明:通过改变膳食中FA的种类可抑制肝脂变细胞的进一步损伤,不会促进该病的进一步恶化。第4章通过检测膳食中FA的种类对人肝脂变细胞免疫调控的影响,探究促进NAFLD到进一步肝炎的发病机制。通过对NO和iNOS测定分析表明:MCFA组与NR组对比不会引起NO和iNOS水平增加,LCFA亦不会引起NO和iNOS的显著增加。细胞免疫分析表明:MCFA不能促进IL-6、IL-1-β和TNF-α经典免疫因子的分泌,而LCFA具有类似LPS的作用,能显著提高上述免疫因子的产生。本章结果表明:FA引起肝脂变细胞的凋亡不依赖于NO和iNOS途径,且LCFA能强烈引起细胞的免疫应答,摄入含MCFA的油脂对NAFLD的进一步恶化为NASH起着积极的作用。第5章脂质在肝脏中过量沉积会引起肝细胞功能的紊乱,进而促进脂肪肝的形成危害机体的健康,但引起脂滴在肝细胞里聚集的机制仍未能得到明确的解释。本章探究MCFA对NAFLD细胞中脂质代谢的影响,试图寻找日常饮食中脂肪的替代品。油红O染色和TG检测实验暗示:与LCFA(油酸,C18:1)对比,MCFA可显著降低肝脂变LO2细胞内脂滴的积累,具有类似吉非罗齐药物的降脂效果。RT-PCR和WB的研究结果表明:MCFA通过下调LPL、FAS、ACC、LXR-α、CD36和SREBP-1脂肪生成基因的在肝脂变细胞内的表达,同时上调ATGL和HSL加速脂肪氧化分解基因的表达,从而使细胞内TG的聚集得到改善,最终使肝细胞的脂肪变程度得到缓和。第6章结论与展望部分对MCFA对NAFLD细胞模型细胞凋亡、抗氧化体系、免疫调控和脂质代谢的分子作用机制进行了归纳,对后续研究工作进行了展望。综上所述,膳食中油脂与NAFLD细胞模型的相互用的分子机制仍需进一步探究。本文从NAFLD细胞模型的建立,以及MCFA对该模型中细胞的活性与凋亡、抗氧化体系、脂质沉淀和免疫反应的生物学作用,并从细胞和分子水平探讨了 MCFA对该模型脂质代谢和免疫调控相关机制。本论文对肥胖人群合理选择食物中FA的种类具有肯定的指导作用,为NAFLD的预防及治疗提供了科学依据。
[Abstract]:With the improvement of people's living standards and the decrease of physical activities, the incidence of obesity and related metabolic diseases has increased significantly, and then the health of the body has been damaged. In this paper, we used combinatorial biology technology to establish the cell model of nonalcoholic fatty liver disease (NAFLD). Fatty acid (FA) was used to investigate the molecular mechanism of apoptosis, oxidative stress, immune regulation and lipid metabolism in NAFLD. Cell viability assay, LDH cell necrosis assay, Hoechst 33342 staining for cell apoptosis assay, oil red O staining and triglyceride (TG) quantitative assay, and scanning electron microscopy for cell membrane damage detection were used to establish NALD model (maximum lipid precipitation and low cytotoxicity) in accordance with the law of human natural diseases. Exploration. The expression of immune factors in diet was investigated by Annexin V-FITC/PI double staining flow cytometry (FCM) and Hoechst 33342/PI fluorescence microscopy, antioxidant and oxidative stress assays, differential proteomics, WB and ELISA combined with fluorescence quantitative PCR. Effects of A species on apoptosis, immune regulation and oxidative stress in human hepatic steatosis cells and the mechanism of promoting NAFLD to further liver injury were investigated. MTT cell activity assay, oil red O staining, TG qualitative and quantitative assay, fluorescence quantitative PCR of lipid metabolism genotype, ELISA and WB were used to study the effect of MCFA on NAFLD. The main results of this study are summarized as follows: In the first chapter, the biological characteristics of MCFA and the research progress of NAFLD are reviewed respectively, and the molecular mechanism of NAFLD is prospected. Advances in the study of NAFLD include the pathogenesis, establishment of models, apoptosis, oxidative stress, immune regulation and related lipid metabolism signaling pathways. Chapter 2 provides the best conditions for the establishment of NAFLD models in accordance with the laws of human natural diseases. The effects of mixed FA (oleic acid: palmitic acid = 2:1) and single FA (oleic acid) on the proliferation and apoptosis of L02 and HepG2 hepatocytes were investigated by MTT and LDH mutual validation. The results showed that the toxicity of FA on both L02 and HepG2 hepatocytes was time-and concentration-dependent. When the concentration of FA was lower than 400 mu M, there was no effect on the cell viability, and L. The results of Hoechst staining showed that the degree of apoptosis was positively correlated with the concentration of FA. When the concentration was less than 400 mu M, no apoptosis was observed compared with the control group. When the concentration was raised to 800 mu M, the apoptosis could be induced. Qualitative observation of lipid droplets and cell morphology by oil red O staining and quantitative determination of intracellular TG by phosphoglycerol oxidase showed that FA mixture was more suitable for establishing hepatic steatosis cell model than single FA. When mixed FA and LO2 cells incubated for 24 hours and the final concentration was 200 mu M, it reached Chapter 3 investigates the effects of dietary FA on apoptosis and oxidative stress in human hepatic steatosis cells, and explores the mechanism of promoting NAFLD to further liver injury. Compared with LCFA, MCFA can significantly reduce early hepatic steatosis. The results of oxidation analysis showed that LCFA could inhibit the production of antioxidant enzymes by protein two-dimensional electrophoresis and secondary mass spectrometry; furthermore, the determination of SOD, MDA, GSH and ROS also showed that MCFA did not produce oxidative stress, but LC did. The results showed that FA could inhibit the further damage of hepatic steatosis cells by changing the types of FA in diet, and could not promote the further deterioration of the disease. The results of NO and iNOS assay showed that the levels of NO and iNOS were not increased in MCFA group compared with NR group, and the levels of NO and iNOS were not significantly increased in LCFA group. The results of this chapter show that FA-induced apoptosis of hepatic steatosis cells does not depend on NO and iNOS pathways, and LCFA can strongly induce cellular immune response. The intake of MCFA-containing lipids plays a positive role in the further deterioration of NAFLD to NASH. Chapter 5 Excessive lipid deposition in the liver can lead to the disorder of hepatocyte function, thereby promoting fatty liver. This chapter explores the effects of MCFA on lipid metabolism in NAFLD cells and attempts to find alternatives to fat in the diet. Oil red O staining and TG assays suggest that compared with LCFA (oleic acid, C18:1), MCFA can significantly reduce liver steatosis L. The results of RT-PCR and WB showed that MCFA could down-regulate the expression of LPL, FAS, ACC, LXR-alpha, CD36 and SREBP-1 adipogenesis genes in hepatic steatosis cells by down-regulating the expression of LPL, FAS, ACC, LXR-alpha, CD36 and SREBP-1 adipogenesis genes, and up-regulating the expression of ATGL and HSL in hepatic steatosis cells. Chapter 6 Conclusion and prospect summarize the molecular mechanism of MCFA on apoptosis, antioxidant system, immune regulation and lipid metabolism of NAFLD cell model, and prospect the follow-up research. In this paper, the establishment of NAFLD cell model and the biological effects of MCFA on cell viability and apoptosis, antioxidant system, lipid precipitation and immune response were studied. The mechanism of lipid metabolism and immune regulation by MCFA was discussed at cellular and molecular levels. Rational selection of FA types in food for obese people has a positive guiding role, which provides a scientific basis for the prevention and treatment of NAFLD.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS221;TS201.4

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