草鱼脂肪细胞发育及HUFA对其影响的研究

发布时间：2018-03-22 18:09

本文选题：草鱼　切入点：脂肪细胞　出处：《西北农林科技大学》2015年博士论文　论文类型：学位论文

【摘要】：脂肪组织是动物体内最大的能量贮存场所,在调控机体的能量平衡中发挥着重要作用。在动物生产中,脂肪组织发育失衡往往引发诸多问题,如造成动物疾病、降低动物产品品质,从而影响养殖效益。脂肪组织主要由脂肪细胞构成,脂肪细胞的发育状况直接决定着脂肪组织的生物性特性及其功能,并影响机体的体脂沉积。因此,阐明脂肪细胞发育机理将为确保动物代谢平衡、改善体脂沉积状况的研究提供重要的理论依据。已有学者研究了脂肪细胞在真鲷和斑马鱼个体发育过程中的发生情况,建立了包括草鱼在内的7种鱼类脂肪细胞的体外培养体系,对鱼类脂肪细胞发育规律进行了一定的研究。但是,草鱼脂肪细胞在体发生的时间及部位,以及其发育过程中的分子事件以及涉及的重要细胞器(如线粒体)的变化情况尚未见报道。高不饱和脂肪酸(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)可通过促进脂质分解、抑制脂质生成和脂肪酸氧化而影响脂肪细胞的发育、降低脂质蓄积,但是其作用机制尚不清楚。本论文旨在研究草鱼脂肪细胞的发育情况,并探讨HUFA影响草鱼脂肪细胞发育(分化及水解)的作用及机制。论文首先采用组织学方法制作石蜡和冰冻切片,并进行HE染色、油红O和BODIPY荧光染色,观测了脂肪细胞在草鱼个体发育过程中出现的时间及部位,而后利用q PCR技术检测了脂质分化相关基因如PPARγ、C/EBPα等在脂肪细胞形成过程中的时序表达情况,用免疫组织化学方法确定PPARγ蛋白的表达部位及时间点;然后,利用体外培养体系,研究了草鱼前体脂肪细胞发育过程中的分化特征,采用电镜切片观察了细胞分化过程中胞内脂滴形态和数量变化并观测了线粒体的数量变化,采用试剂盒检测了分化过程中线粒体蛋白的含量变化,实时定量PCR技术检测了细胞分化及线粒体发育相关基因的表达。此外,为了研究HUFA对草鱼脂肪细胞分化的影响,以DHA处理分化过程中的细胞,利用转录组测序技术分析了处理前后表达状况出现差异的基因,发现DHA可以影响到细胞中调控细胞分化的信号通路;其次,为了探究Wnt/β-catenin信号通路在DHA影响脂肪细胞分化中的作用,以DHA和β-catenin的抑制剂FH535分别或者共同处理分化的脂肪细胞,检测细胞分化过程中脂质含量、基因及蛋白的表达变化;最后,分别研究了HUFA(DHA和EPA)在离体及在体条件下对草鱼成熟脂肪细胞水解的影响,离体方面分离草鱼成熟脂肪细胞以HUFA处理后分别检测了甘油释放量及基因表达变化,在体方面,以富含HUFA饲喂草鱼95天,随后分离成熟脂肪细胞测定其在离体条件下的基础水解情况并检测了脂肪组织基因表达情况。获得的主要研究结果如下:1.在体研究结果表明,草鱼腹腔脂肪细胞在受精卵开始发育16天后于仔鱼肝胰脏周边出现,随后逐渐分布至肠系膜周围,形成脂肪组织;脂肪组织发育初期以脂肪细胞个体数目增加为主,该过程与脂质合成相关基因的表达相协调;2.离体研究发现,草鱼前体脂肪细胞分化过程中,细胞脂质含量及脂滴数量、线粒体数量和蛋白量含量、脂质生成及线粒体发育相关基因的表达水平均显著升高,表明线粒体在脂肪细胞分化过程中发挥着一定的作用;3.DHA作用于分化阶段草鱼前体脂肪细胞2天可促进脂肪细胞脂质蓄积,处理7天作用相反,且脂质生成相关基因的表达同步变化。高通量测序结果进一步表明,处理2天上调脂质生成相关基因的表达,处理7天下调脂质生成相关基因的表达,并促进Wnt/β-catenin信号通路中β-catenin的表达,表明Wnt信号通路可能在DHA影响脂肪细胞分化中发挥着重要作用;4.DHA处理可显著抑制脂肪细胞的脂质蓄积,而同时抑制β-catenin活性可缓解DHA对脂肪细胞脂质蓄积的抑制作用。实时定量结果表明DHA还可激活脂肪细胞分化过程中β-catenin及其下游细胞增殖相关基因如c-myc、c-jun、和cyc D1的表达,抑制β-catenin则可以抑制DHA对β-catenin、cyc D1和c-myc的促进作用。另外,DHA还可促进β-catenin蛋白的表达。表明DHA可能通过激活Wnt/β-catenin通路,影响细胞周期,进而抑制脂肪细胞分化;5.HUFA体外处理可促进草鱼成熟脂肪细胞甘油和游离脂肪酸的释放,促进细胞ATGL和HSL等水解相关基因的表达;在体饲喂HUFA可促进草鱼成熟脂肪细胞的水解能力,并促进脂肪组织ATGL和HSL等水解相关基因的表达。表明HUFA可促进草鱼脂肪细胞水解。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S917.4

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