ADAMTS2和ADAMTS4基因对牛前体脂肪细胞分化及脂质沉积的影响

发布时间：2018-05-01 21:37

本文选题：ADAMTS2基因 + ADAMTS4基因　；参考：《河南科技大学》2017年硕士论文

【摘要】：脂肪组织对机体能量代谢和平衡具有重要作用,同时对动物的肉质产生很大影响。牛肉的嫩度、风味和多汁性主要受肌内脂肪含量的影响。脂肪细胞是由前体脂肪细胞分化而来的,此过程受激素和生长因子的调控,这些激素或生长因子的改变会影响前体脂肪细胞的分化。研究表明,ADAMTS2基因是原胶原转变成胶原过程的剪切酶,它通过对胶原蛋白的生物合成调控脂质的沉积。ADAMTS4基因是牛肉形成大理石纹状的重要基因之一。由于ADAMTS2和ADAMTS4基因都能对脂肪生成进行调控,从而推测ADAMTS2和ADAMTS4基因的表达与前体脂肪细胞分化和肌肉内脂肪的沉积有关。因此,本文以牛前体脂肪细胞为研究对象,分析ADAMTS2或ADAMTS4基因对脂质沉积的影响,来获得ADAMTS2和ADAMTS4基因在牛前体脂肪细胞分化过程中的作用。本研究首先采取犊牛皮下脂肪组织进行体外分离和培养。其次将合成的siRNA通过lipo-2000转染至前体脂肪细胞中,特异性敲减ADAMTS2和ADAMTS4基因的表达,通过q RT-PCR方法检测与脂肪细胞分化相关基因mRNA表达量变化情况,用胰岛素等对转染后的细胞进行诱导,通过HE染色、MASSON染色和扫描电镜方法检测脂质沉积情况,Western-Blot来检测转染后蛋白表达情况。结果表明,敲减ADAMTS2基因后,与脂肪细胞分化相关基因PPAR、DGAT2、COL1A1、COL3A1、ADAMTS2、ADAMTS4和Aggrecan的mRNA表达量均降低,而与脂肪细胞分化相关基因EBP和COL2A1的mRNA表达量无明显变化。敲减ADAMTS4基因后,与脂肪细胞分化相关基因PPAR、DGAT2、COL3A1、ADAMTS2、ADAMTS4和Aggrecan的mRNA表达量均降低,而与脂肪细胞分化相关基因COL1A1、EBP和COL2A1基因m RNA表达量无明显变化。敲减ADAMTS2或ADAMTS4基因并经过诱导后,实验组脂质量明显比对照组少。Western-Blot分析表明,ADAMTS2和ADAMTS4蛋白在siRNA转染后,蛋白表达量均降低。说明ADAMTS2和ADAMTS4基因均与脂肪细胞分化相关,二者对前体脂肪细胞分化作用机制不同,本研究从形态学和分子调控机制方面对ADAMTS2和ADAMTS4基因在牛前体脂肪细胞分化中的作用进行了分析,这有助于揭示脂肪分化的分子机制,为提高牛肉品质及良种选育提供理论依据。
[Abstract]:Adipose tissue plays an important role in energy metabolism and balance. The tenderness, flavor and juicy of beef are mainly affected by the content of intramuscular fat. Adipocytes are derived from precursor adipocytes, which are regulated by hormones and growth factors, which affect the differentiation of precursor adipocytes. It is shown that ADAMTS2 gene is a shearing enzyme in the process of procollagen conversion to collagen. ADAMTS4 gene is one of the important genes for marbling formation of beef through the regulation of lipid deposition through the biosynthesis of collagen. Since both ADAMTS2 and ADAMTS4 genes can regulate adipogenesis, it is assumed that the expression of ADAMTS2 and ADAMTS4 genes is related to the differentiation of precursor adipocytes and the deposition of fat in muscle. Therefore, the effect of ADAMTS2 or ADAMTS4 gene on lipid deposition was analyzed in order to obtain the role of ADAMTS2 and ADAMTS4 genes in the differentiation of bovine precursor adipocytes. In this study, calf subcutaneous adipose tissue was firstly isolated and cultured in vitro. Secondly, the synthesized siRNA was transfected into the precursor adipocytes by lipo-2000, and the expression of ADAMTS2 and ADAMTS4 genes was reduced by specific knockout. The expression of mRNA related to adipocyte differentiation was detected by Q RT-PCR method. The transfected cells were induced by insulin, and the protein expression was detected by HE staining and scanning electron microscopy (SEM), and the lipid deposition was detected by Western-Blot. The results showed that after knockout of ADAMTS2 gene, the mRNA expression of adipocyte differentiation related gene PPARN DGAT2COL1A1, COL3A1, ADAMTS2, ADAMTS4 and Aggrecan decreased, but the mRNA expression of adipocyte differentiation related genes EBP and COL2A1 did not change significantly. After knockout of ADAMTS4 gene, the expression of mRNA and Aggrecan in adipocyte differentiation related gene PPARN DGA2A1C, ADAMTS2, ADAMTS4 and ADAMTS4 were all decreased, but the expression of COL1A1EBP and COL2A1 gene m RNA were not significantly changed in adipocyte differentiation related gene (COL1A1EBP) and COL2A1 gene (m RNA), but not in adipocyte differentiation related gene (COL1A1EBP and COL2A1 gene). After knockout and induction of ADAMTS2 or ADAMTS4 gene, the lipid mass of the experimental group was significantly lower than that of the control group. Western-Blot analysis showed that the expression of ADAMTS2 and ADAMTS4 protein decreased after siRNA transfection. Both ADAMTS2 and ADAMTS4 genes were related to adipocyte differentiation, and the mechanism of differentiation of preadipocytes was different between them. In this study, the effects of ADAMTS2 and ADAMTS4 genes on the differentiation of bovine precursor adipocytes were analyzed in terms of morphology and molecular regulation mechanism, which was helpful to reveal the molecular mechanism of adipocyte differentiation and to provide theoretical basis for improving beef quality and breeding of improved breeds.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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