發(fā)布時(shí)間：2018-08-26 11:08

【摘要】：隨著人們物質(zhì)生活水平的提高以及體育活動(dòng)的減少,導(dǎo)致肥胖及其相關(guān)代謝疾病的發(fā)病率顯著增加,繼而損傷機(jī)體的健康。所以,近年來(lái)尋求一種綠色高效的減肥手段一貫被研究者所重視。然而膳食油脂作為引起體內(nèi)脂質(zhì)沉淀的主要原因,對(duì)機(jī)體的生物學(xué)作用和內(nèi)在分子機(jī)制不甚清楚。本論文采用組合生物學(xué)技術(shù),對(duì)非酒精性脂肪肝病(NAFLD)細(xì)胞模型的建立、脂肪酸(FA)對(duì)該模型細(xì)胞凋亡、氧化壓力、免疫調(diào)控和脂質(zhì)代謝的分子作用機(jī)制進(jìn)行了非常全面的探討。采用MTT細(xì)胞活性檢測(cè)法、LDH細(xì)胞壞死檢測(cè)法、Hoechst 33342染色對(duì)細(xì)胞凋亡測(cè)定法、油紅O染色法和甘油三酯(TG)定量測(cè)定法、掃描電鏡對(duì)細(xì)胞膜損傷程度檢測(cè)法對(duì)建立符合人類自然病規(guī)律的NAFLD模型(最大脂質(zhì)沉淀和較低的細(xì)胞毒性)的條件進(jìn)行了探索。采用檢測(cè)細(xì)胞凋亡的Annexin V-FITC/PI雙染色流式細(xì)胞儀法(FCM)和Hoechst33342/PI熒光顯微鏡法、細(xì)胞的抗氧化及氧化壓力的酶標(biāo)儀測(cè)定法、差異蛋白測(cè)定的蛋白組學(xué)法、WB以及ELISA結(jié)合熒光定量PCR技術(shù)考察了免疫因子的表達(dá),研究膳食中FA的種類對(duì)人肝脂變細(xì)胞凋亡、免疫調(diào)控和氧化壓力的影響,探究促進(jìn)NAFLD到進(jìn)一步肝損傷的機(jī)制。采用MTT細(xì)胞活性檢測(cè)法、油紅O染色和TG定性定量測(cè)定法以及脂代謝基因型熒光定量PCR、ELISA與WB相結(jié)合的方法,對(duì)中碳鏈脂肪酸(MCFA)對(duì)NAFLD細(xì)胞脂滴的聚集及相關(guān)脂代謝的分子作用機(jī)制進(jìn)行了全面的探究。本研究的主要結(jié)果總結(jié)如下:第1章緒論部分對(duì)MCFA的生物學(xué)特性和NAFLD的研究進(jìn)展分別做了綜述,并對(duì)引起NAFLD的分子作用機(jī)制作出了展望。FA生物學(xué)特性,包括FA的代謝特點(diǎn)、生理功能、安全性和潛在的局限性的研究進(jìn)展。NAFLD研究部分包括該病發(fā)病的原因、模型的建立、引起的細(xì)胞凋亡、氧化壓力、免疫調(diào)控和相關(guān)脂代謝信號(hào)通路的研究進(jìn)展。第2章對(duì)建立符合人類自然疾病規(guī)律的NAFLD模型的最適條件進(jìn)行了探索。通過(guò)MTT和LDH相互驗(yàn)證的實(shí)驗(yàn)方法考察混合FA(油酸:棕櫚酸=2:1)和單一FA(油酸)對(duì)L02和HepG2兩種肝細(xì)胞的增殖與凋亡的影響,結(jié)果發(fā)現(xiàn)FA對(duì)細(xì)胞的毒性均產(chǎn)生濃度和時(shí)間依賴性,當(dāng)濃度低于400μM時(shí),不會(huì)對(duì)細(xì)胞的活性存在影響,且LO2細(xì)胞對(duì)FA的毒性影響較HepG2肝細(xì)胞更加敏感。進(jìn)一步通過(guò)Hoechst染色法對(duì)細(xì)胞凋亡的檢測(cè)結(jié)果說(shuō)明細(xì)胞凋亡的程度與FA的濃度呈正相關(guān),在濃度小于400 μM時(shí),與對(duì)照組相比均不會(huì)明顯引起細(xì)胞的凋亡;濃度升高到800 μM時(shí),均可引起細(xì)胞的毒性。通過(guò)油紅O染色定性觀察細(xì)胞內(nèi)的脂滴及細(xì)胞的形態(tài),磷酸甘油氧化酶法定量對(duì)胞里TG含量的測(cè)定,結(jié)果表明FA混合物相對(duì)于單一 FA更適合用來(lái)建立肝脂變細(xì)胞模型;當(dāng)混合FA與LO2細(xì)胞孵育時(shí)間為24 h,作用終濃度為200μM 時(shí),到達(dá)最高的脂質(zhì)沉淀且造成的肝細(xì)胞慢性損傷和NAFLD的慢性肝脂肪變接近。第3章通過(guò)研究膳食中FA的種類對(duì)人肝脂變細(xì)胞凋亡和氧化壓力的影響,探究促進(jìn)NAFLD到進(jìn)一步肝損傷的機(jī)制。通過(guò)對(duì)細(xì)胞凋亡分析表明:同LCFA對(duì)比,MCFA能顯著降低早、中、晚期凋亡細(xì)胞和壞死細(xì)胞的百分比,同時(shí)抑制Caspase3和Caspase9酶的活性。氧化分析結(jié)果表明:采用蛋白雙向電泳及二級(jí)質(zhì)譜方法研究發(fā)現(xiàn)LCFA能抑制抗氧化酶的產(chǎn)生;進(jìn)一步通過(guò)對(duì)SOD、MDA、GSH和ROS的測(cè)定也發(fā)現(xiàn)MCFA不會(huì)產(chǎn)生氧化壓力,而LCFA會(huì)對(duì)細(xì)胞產(chǎn)生氧化壓力。本章結(jié)果表明:通過(guò)改變膳食中FA的種類可抑制肝脂變細(xì)胞的進(jìn)一步損傷,不會(huì)促進(jìn)該病的進(jìn)一步惡化。第4章通過(guò)檢測(cè)膳食中FA的種類對(duì)人肝脂變細(xì)胞免疫調(diào)控的影響,探究促進(jìn)NAFLD到進(jìn)一步肝炎的發(fā)病機(jī)制。通過(guò)對(duì)NO和iNOS測(cè)定分析表明:MCFA組與NR組對(duì)比不會(huì)引起NO和iNOS水平增加,LCFA亦不會(huì)引起NO和iNOS的顯著增加。細(xì)胞免疫分析表明:MCFA不能促進(jìn)IL-6、IL-1-β和TNF-α經(jīng)典免疫因子的分泌,而LCFA具有類似LPS的作用,能顯著提高上述免疫因子的產(chǎn)生。本章結(jié)果表明:FA引起肝脂變細(xì)胞的凋亡不依賴于NO和iNOS途徑,且LCFA能強(qiáng)烈引起細(xì)胞的免疫應(yīng)答,攝入含MCFA的油脂對(duì)NAFLD的進(jìn)一步惡化為NASH起著積極的作用。第5章脂質(zhì)在肝臟中過(guò)量沉積會(huì)引起肝細(xì)胞功能的紊亂,進(jìn)而促進(jìn)脂肪肝的形成危害機(jī)體的健康,但引起脂滴在肝細(xì)胞里聚集的機(jī)制仍未能得到明確的解釋。本章探究MCFA對(duì)NAFLD細(xì)胞中脂質(zhì)代謝的影響,試圖尋找日常飲食中脂肪的替代品。油紅O染色和TG檢測(cè)實(shí)驗(yàn)暗示:與LCFA(油酸,C18:1)對(duì)比,MCFA可顯著降低肝脂變LO2細(xì)胞內(nèi)脂滴的積累,具有類似吉非羅齊藥物的降脂效果。RT-PCR和WB的研究結(jié)果表明:MCFA通過(guò)下調(diào)LPL、FAS、ACC、LXR-α、CD36和SREBP-1脂肪生成基因的在肝脂變細(xì)胞內(nèi)的表達(dá),同時(shí)上調(diào)ATGL和HSL加速脂肪氧化分解基因的表達(dá),從而使細(xì)胞內(nèi)TG的聚集得到改善,最終使肝細(xì)胞的脂肪變程度得到緩和。第6章結(jié)論與展望部分對(duì)MCFA對(duì)NAFLD細(xì)胞模型細(xì)胞凋亡、抗氧化體系、免疫調(diào)控和脂質(zhì)代謝的分子作用機(jī)制進(jìn)行了歸納,對(duì)后續(xù)研究工作進(jìn)行了展望。綜上所述,膳食中油脂與NAFLD細(xì)胞模型的相互用的分子機(jī)制仍需進(jìn)一步探究。本文從NAFLD細(xì)胞模型的建立,以及MCFA對(duì)該模型中細(xì)胞的活性與凋亡、抗氧化體系、脂質(zhì)沉淀和免疫反應(yīng)的生物學(xué)作用,并從細(xì)胞和分子水平探討了 MCFA對(duì)該模型脂質(zhì)代謝和免疫調(diào)控相關(guān)機(jī)制。本論文對(duì)肥胖人群合理選擇食物中FA的種類具有肯定的指導(dǎo)作用,為NAFLD的預(yù)防及治療提供了科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS221;TS201.4

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