肌联素对猪肌内脂肪细胞分化及脂肪酸利用的调控研究

发布时间：2018-08-02 20:11

【摘要】：本研究通过分离培养猪骨骼肌卫星细胞和肌内前体脂肪细胞,利用骨骼肌细胞和肌内脂肪细胞间接共培养、重组肌联素处理肌内脂肪细胞等技术手段,研究肌肉细胞对肌内脂肪细胞分化,肌联素对肌内脂肪细胞分化、脂肪细胞中脂肪酸摄取、转运和利用的影响。试验获得以下结果:1.为了研究猪骨骼肌细胞对肌内前体脂肪细胞分化的影响,利用猪骨骼肌卫星细胞和肌内前体脂肪细胞共培养,分别诱导分化,检测肌内脂肪细胞分化水平、脂肪细胞分化标志基因的表达以及脂代谢关键酶的表达。结果表明:与肌内脂肪细胞单独培养相比,共培养体系中1)肌内脂肪细胞脂滴数量和脂滴面积显著减少(P0.05);2)肌内脂肪细胞增殖分化标志基因PPARγ和C/EBPα的表达量极显著下降(P0.01);3)肌内脂肪细胞ACC、FAS的表达水平极显著下降(P0.01);4)骨骼肌细胞内肌联素的基因表达水平随时间而极显著地增加。2.为了研究重组肌联素对肌内脂肪细胞利用外源游离脂肪酸的调控作用,试验采用重组肌联素和脂肪酸单独或共同处理猪肌内脂肪细胞,研究肌联素对肌内脂肪细胞分化、肌内脂肪细胞摄入外周游离脂肪酸的能力、脂质合成与分解代谢以及脂肪细胞线粒体脂肪酸氧化的影响,检测肌内脂肪细胞培养基中游离脂肪酸的含量、肌内脂肪细胞脂肪酸转运相关基因和蛋白的表达及其作用的信号通路。结果表明:1)肌联素单独作用能极显著降低肌内脂肪细胞的脂滴数量和脂滴面积(P0.01),并极显著地促进对游离脂肪酸的吸收(P0.01);2)肌联素单独处理或与棕榈酸共同处理,能显著提高肌内脂肪细胞中脂肪酸转运相关因子FATP1、FABP4的表达水平(P0.05);3)肌联素单独处理或与棕榈酸共同处理后显著提高肌内脂肪细胞HSL、LPL的表达量(P0.05);4)肌联素通过激活p38MAPK信号通路调节脂肪酸转运和脂代谢。3.为了研究重组肌联素对猪肌内脂肪细胞中脂肪酸氧化的影响,试验采用重组肌联素处理猪肌内脂肪细胞,检测线粒体中与脂肪酸氧化相关的标志因子和蛋白的表达。结果表明:1)肌联素能显著提高肌内脂肪细胞线粒体中脂肪酸氧化标志因子TFAM、UCP1的表达水平(P0.05);2)肌联素显著提高了肌内脂肪细胞线粒体中氧化呼吸链标志蛋白复合物Ⅰ的表达水平(P0.05)。综上所述,本研究证明猪骨骼肌细胞对肌内前体脂肪细胞分化具有抑制作用;肌联素能抑制肌内前体脂肪细胞分化;肌联素可促进猪肌内脂肪细胞对外源脂肪酸的摄取及转运;肌联素对猪肌内脂肪细胞线粒体的脂肪酸氧化具有促进作用。
[Abstract]:In this study, porcine skeletal muscle satellite cells and intramuscular precursor adipocytes were isolated and cultured. Indirect co-culture of skeletal muscle cells and intramuscular adipocytes was carried out. To study the effects of muscle cells on intramuscular adipocyte differentiation, myopectin on intramuscular adipocyte differentiation, fatty acid uptake, transport and utilization in adipocytes. The results of the experiment are as follows: 1. In order to study the effect of porcine skeletal muscle cells on the differentiation of intramuscular preadipocytes, porcine skeletal muscle satellite cells and intramuscular precursor adipocytes were co-cultured to induce differentiation and detect the differentiation level of intramuscular adipocytes. The expression of adipocyte differentiation marker gene and lipid metabolism key enzymes. The results showed that compared with intramuscular adipocyte culture alone, In co-culture system 1) the number and area of lipid droplets in intramuscular adipocytes were significantly decreased (P0.05) and the expression of C/EBP 伪 and PPAR 纬 were significantly decreased (P0.01) 3) the expression level of ACC-FAS in intramuscular adipocytes was extremely significant. (P0.01) 4) the gene expression level of sarcopectin in skeletal muscle cells increased significantly with time. In order to study the effect of recombinant sarcopectin on the utilization of exogenous free fatty acids in intramuscular adipocytes, the effects of recombinant myonectin and fatty acids on the differentiation of intramuscular adipocytes were studied. The ability of intramuscular adipocytes to ingest peripheral free fatty acids, the effects of lipid synthesis and catabolism, and the oxidation of fatty acids in mitochondria of adipocytes were measured. Expression of fatty acid transporter related genes and proteins in muscle adipocytes and signal pathway of their action. The results showed that the concentration of lipid droplets and lipid drop area (P0.01) in muscle adipocytes were significantly decreased by the action of 1: 1), and the absorption of free fatty acids (P0.01) 2) was promoted by the treatment of myopectin alone or in combination with palmitic acid. The expression level of FABP4 in intramuscular adipocytes was significantly increased (P0.05). (P0.05) the expression of HSL-LPL in intramuscular adipocytes was significantly increased (P0.05). (P0.05) the expression of HSL-LPL in intramuscular adipocytes was significantly increased (P0.05) by activation of p38MAPK signal by myopectin alone or in combination with palmitic acid. Pathway No.3 regulates fatty acid transport and lipid metabolism. In order to study the effect of recombinant sarcopectin on fatty acid oxidation in porcine intramuscular adipocytes, the expression of markers and proteins related to fatty acid oxidation in mitochondria was detected by using recombinant myosin to treat porcine intramuscular adipocytes. The results showed that the expression of TFAM- UCP1 in mitochondria of intramuscular adipocytes was significantly increased (P0.05). The expression of oxidative respiratory chain marker protein complex I in mitochondria of intramuscular adipocytes was significantly increased (P0.05). To sum up, this study demonstrated that porcine skeletal muscle cells can inhibit the differentiation of intramuscular precursor adipocytes, that sarcopectin can inhibit the differentiation of intramuscular preadipocytes, and that sarcopectin can promote the uptake and transport of exogenous fatty acids by porcine muscle adipocytes. Adiponectin promotes fatty acid oxidation in mitochondria of porcine intramuscular adipocytes.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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