發(fā)布時(shí)間：2018-03-22 18:09

  本文選題：草魚　切入點(diǎn)：脂肪細(xì)胞　出處：《西北農(nóng)林科技大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：脂肪組織是動(dòng)物體內(nèi)最大的能量貯存場(chǎng)所,在調(diào)控機(jī)體的能量平衡中發(fā)揮著重要作用。在動(dòng)物生產(chǎn)中,脂肪組織發(fā)育失衡往往引發(fā)諸多問題,如造成動(dòng)物疾病、降低動(dòng)物產(chǎn)品品質(zhì),從而影響?zhàn)B殖效益。脂肪組織主要由脂肪細(xì)胞構(gòu)成,脂肪細(xì)胞的發(fā)育狀況直接決定著脂肪組織的生物性特性及其功能,并影響機(jī)體的體脂沉積。因此,闡明脂肪細(xì)胞發(fā)育機(jī)理將為確保動(dòng)物代謝平衡、改善體脂沉積狀況的研究提供重要的理論依據(jù)。已有學(xué)者研究了脂肪細(xì)胞在真鯛和斑馬魚個(gè)體發(fā)育過程中的發(fā)生情況,建立了包括草魚在內(nèi)的7種魚類脂肪細(xì)胞的體外培養(yǎng)體系,對(duì)魚類脂肪細(xì)胞發(fā)育規(guī)律進(jìn)行了一定的研究。但是,草魚脂肪細(xì)胞在體發(fā)生的時(shí)間及部位,以及其發(fā)育過程中的分子事件以及涉及的重要細(xì)胞器(如線粒體)的變化情況尚未見報(bào)道。高不飽和脂肪酸(highly unsaturated fatty acid,HUFA)如二十碳五烯酸(Eicosapentaenoic acid,EPA)和二十二碳六烯酸(Docosahexaenoic acid,DHA)可通過促進(jìn)脂質(zhì)分解、抑制脂質(zhì)生成和脂肪酸氧化而影響脂肪細(xì)胞的發(fā)育、降低脂質(zhì)蓄積,但是其作用機(jī)制尚不清楚。本論文旨在研究草魚脂肪細(xì)胞的發(fā)育情況,并探討HUFA影響草魚脂肪細(xì)胞發(fā)育(分化及水解)的作用及機(jī)制。論文首先采用組織學(xué)方法制作石蠟和冰凍切片,并進(jìn)行HE染色、油紅O和BODIPY熒光染色,觀測(cè)了脂肪細(xì)胞在草魚個(gè)體發(fā)育過程中出現(xiàn)的時(shí)間及部位,而后利用q PCR技術(shù)檢測(cè)了脂質(zhì)分化相關(guān)基因如PPARγ、C/EBPα等在脂肪細(xì)胞形成過程中的時(shí)序表達(dá)情況,用免疫組織化學(xué)方法確定PPARγ蛋白的表達(dá)部位及時(shí)間點(diǎn);然后,利用體外培養(yǎng)體系,研究了草魚前體脂肪細(xì)胞發(fā)育過程中的分化特征,采用電鏡切片觀察了細(xì)胞分化過程中胞內(nèi)脂滴形態(tài)和數(shù)量變化并觀測(cè)了線粒體的數(shù)量變化,采用試劑盒檢測(cè)了分化過程中線粒體蛋白的含量變化,實(shí)時(shí)定量PCR技術(shù)檢測(cè)了細(xì)胞分化及線粒體發(fā)育相關(guān)基因的表達(dá)。此外,為了研究HUFA對(duì)草魚脂肪細(xì)胞分化的影響,以DHA處理分化過程中的細(xì)胞,利用轉(zhuǎn)錄組測(cè)序技術(shù)分析了處理前后表達(dá)狀況出現(xiàn)差異的基因,發(fā)現(xiàn)DHA可以影響到細(xì)胞中調(diào)控細(xì)胞分化的信號(hào)通路;其次,為了探究Wnt/β-catenin信號(hào)通路在DHA影響脂肪細(xì)胞分化中的作用,以DHA和β-catenin的抑制劑FH535分別或者共同處理分化的脂肪細(xì)胞,檢測(cè)細(xì)胞分化過程中脂質(zhì)含量、基因及蛋白的表達(dá)變化;最后,分別研究了HUFA(DHA和EPA)在離體及在體條件下對(duì)草魚成熟脂肪細(xì)胞水解的影響,離體方面分離草魚成熟脂肪細(xì)胞以HUFA處理后分別檢測(cè)了甘油釋放量及基因表達(dá)變化,在體方面,以富含HUFA飼喂草魚95天,隨后分離成熟脂肪細(xì)胞測(cè)定其在離體條件下的基礎(chǔ)水解情況并檢測(cè)了脂肪組織基因表達(dá)情況。獲得的主要研究結(jié)果如下:1.在體研究結(jié)果表明,草魚腹腔脂肪細(xì)胞在受精卵開始發(fā)育16天后于仔魚肝胰臟周邊出現(xiàn),隨后逐漸分布至腸系膜周圍,形成脂肪組織;脂肪組織發(fā)育初期以脂肪細(xì)胞個(gè)體數(shù)目增加為主,該過程與脂質(zhì)合成相關(guān)基因的表達(dá)相協(xié)調(diào);2.離體研究發(fā)現(xiàn),草魚前體脂肪細(xì)胞分化過程中,細(xì)胞脂質(zhì)含量及脂滴數(shù)量、線粒體數(shù)量和蛋白量含量、脂質(zhì)生成及線粒體發(fā)育相關(guān)基因的表達(dá)水平均顯著升高,表明線粒體在脂肪細(xì)胞分化過程中發(fā)揮著一定的作用;3.DHA作用于分化階段草魚前體脂肪細(xì)胞2天可促進(jìn)脂肪細(xì)胞脂質(zhì)蓄積,處理7天作用相反,且脂質(zhì)生成相關(guān)基因的表達(dá)同步變化。高通量測(cè)序結(jié)果進(jìn)一步表明,處理2天上調(diào)脂質(zhì)生成相關(guān)基因的表達(dá),處理7天下調(diào)脂質(zhì)生成相關(guān)基因的表達(dá),并促進(jìn)Wnt/β-catenin信號(hào)通路中β-catenin的表達(dá),表明Wnt信號(hào)通路可能在DHA影響脂肪細(xì)胞分化中發(fā)揮著重要作用;4.DHA處理可顯著抑制脂肪細(xì)胞的脂質(zhì)蓄積,而同時(shí)抑制β-catenin活性可緩解DHA對(duì)脂肪細(xì)胞脂質(zhì)蓄積的抑制作用。實(shí)時(shí)定量結(jié)果表明DHA還可激活脂肪細(xì)胞分化過程中β-catenin及其下游細(xì)胞增殖相關(guān)基因如c-myc、c-jun、和cyc D1的表達(dá),抑制β-catenin則可以抑制DHA對(duì)β-catenin、cyc D1和c-myc的促進(jìn)作用。另外,DHA還可促進(jìn)β-catenin蛋白的表達(dá)。表明DHA可能通過激活Wnt/β-catenin通路,影響細(xì)胞周期,進(jìn)而抑制脂肪細(xì)胞分化;5.HUFA體外處理可促進(jìn)草魚成熟脂肪細(xì)胞甘油和游離脂肪酸的釋放,促進(jìn)細(xì)胞ATGL和HSL等水解相關(guān)基因的表達(dá);在體飼喂HUFA可促進(jìn)草魚成熟脂肪細(xì)胞的水解能力,并促進(jìn)脂肪組織ATGL和HSL等水解相關(guān)基因的表達(dá)。表明HUFA可促進(jìn)草魚脂肪細(xì)胞水解。
[Abstract]:Adipose tissue is the largest animal used for energy storage, plays an important role in the regulation of energy balance in the body. In animal production, adipose tissue development imbalance often caused many problems, such as the cause of animal diseases, reduce the quality of animal products, thus affecting the breeding efficiency. Adipose tissue is mainly composed of fat cells, fat cells and development status directly determines the biological characteristics and function of adipose tissue, and the effect of body fat deposition. Therefore, to clarify the mechanism of adipocyte development will ensure animal metabolism, provide an important theoretical basis of improving body fat deposition conditions. The scholars have studied the incidence of fat cells in the ontogeny of zebrafish and sea bream the training system has established 7 kinds of fish fat cells including grass carp in vitro on fish fat cell development law of the The study. However, grass carp fat cells in time and position of body, and the molecular events in the development process and the important organelles involved (such as mitochondria) changes have not been reported. High unsaturated fatty acids (highly unsaturated fatty acid, HUFA) as the twenty carbon five (Eicosapentaenoic acid, EPA acid twenty-two) and carbon (Docosahexaenoic acid, DHA six acid) can promote lipolysis, inhibit lipid formation and fatty acid oxidation affect the development of fat cells, reduce lipid accumulation, but its mechanism is still unclear. The aim of this paper is to study the development of grass carp fat cells, and to explore the effect of fat cell Development (grass carp HUFA differentiation and hydrolysis) effect and mechanism. The paper first uses the tissue paraffin and frozen section method, and the HE staining, oil red O and BODIPY fluorescence staining was observed in fat cells The time and location of the grass carp individual development process, and then using the Q PCR technique to detect lipid differentiation related genes such as PPAR gamma, C/EBP alpha formed in the process of sequential expression of fat cells, determine the expression sites and time points PPAR gamma protein by immunohistochemical method; then, using the in vitro culture system, research the differentiation features in the process of grass carp preadipocyte development by electron microscopic observation of the form and the number of lipid droplets changes in intracellular cell differentiation and changes in the number of mitochondria were observed, using the kit to detect the changes of mitochondrial protein content points in the process of real-time quantitative PCR detection of the expression of genes related to cell differentiation and the development of mitochondria. In addition, in order to study the effect of HUFA on grass carp fat cell differentiation, treatment with DHA in the differentiation of cells, analysis of transcriptome sequencing technology use The difference between before and after treatment the expression of DHA gene, that can affect cell signaling pathways regulating cell differentiation; secondly, in order to explore the Wnt/ beta -catenin signal pathway in the effects of DHA in differentiation of adipocytes, DHA and beta -catenin inhibitors of FH535 fat cells respectively or jointly with differentiation, lipid content was detected the process of differentiation, expression of gene and protein; finally, the HUFA (DHA and EPA) were studied in vitro and the effect on the hydrolysis of grass carp mature fat cells in vivo, in vitro separation grass carp mature fat cells in HUFA treatment were detected after glycerol release and gene expression changes in the body. In HUFA, with grass carp for 95 days, followed by isolation of mature adipocytes was measured in vitro of hydrolysis and detected adipose tissue gene expression obtained. The main results are as follows: 1. the research results show that grass carp abdominal fat cells in the zygote after 16 days of development in larval hepatopancreas surrounding, then gradually spread to the surrounding mesenteric adipose tissue formation, the initial increase in fat cells; the number of individual based development of adipose tissue, the expression of genes related to lipid synthesis process and phase coordination; 2. in vitro studies show that grass carp preadipocyte differentiation process, cell lipid content and lipid droplet number, the number of mitochondria and protein content, the expression level of genes related to lipid formation and development of mitochondria were significantly increased, indicating that mitochondria play a role in adipocyte differentiation; the effect of 3.DHA on differentiation stage grass carp preadipocytes 2 days can promote adipocyte lipid accumulation, in contrast to the processing for 7 days, and the production of lipid related genes expression of high synchronous changes. Flux sequencing results further showed that the expression of genes related to processing to generate 2 Heaven regulating lipid, expression of genes related to the generation of 7 days of lipid and the expression of -catenin beta Wnt/ beta -catenin signaling pathway, Wnt signaling pathway may be indicated in DHA adipocytes differentiation plays an important role in the accumulation of lipid 4.DHA treatment; inhibit fat cells, but also inhibit beta -catenin activity can alleviate the inhibitory effect of DHA on adipocyte lipid accumulation. Real time quantitative results show that DHA can be activated during adipocyte differentiation and its downstream -catenin beta cell proliferation related genes such as c-myc, c-jun, CYC and the expression of D1, inhibit beta -catenin can inhibit DHA beta -catenin, cyc D1 and c-myc role. In addition, DHA also can promote the expression of beta -catenin protein. The results indicated that DHA may activate the Wnt/ beta -catenin pathway, affect cell cycle Then, inhibit fat cell differentiation; in vitro 5.HUFA treatment can promote grass carp mature fat cells of glycerol and free fatty acid release, promote the expression of ATGL and HSL genes in vivo hydrolysis; feeding HUFA could accelerate the hydrolysis ability of grass carp mature adipocytes, and promoting the expression of ATGL and HSL in adipose tissue hydrolysis related genes. Grass carp fat cells showed that HUFA can promote the hydrolysis.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S917.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Anna Park;Won Kon Kim;Kwang-Hee Bae;;Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells[J];World Journal of Stem Cells;2014年01期

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本文編號(hào)：1649792