本文選題：ADAMTS2基因 + ADAMTS4基因��； 參考：《河南科技大學》2017年碩士論文

【摘要】：脂肪組織對機體能量代謝和平衡具有重要作用,同時對動物的肉質(zhì)產(chǎn)生很大影響。牛肉的嫩度、風味和多汁性主要受肌內(nèi)脂肪含量的影響。脂肪細胞是由前體脂肪細胞分化而來的,此過程受激素和生長因子的調(diào)控,這些激素或生長因子的改變會影響前體脂肪細胞的分化。研究表明,ADAMTS2基因是原膠原轉(zhuǎn)變成膠原過程的剪切酶,它通過對膠原蛋白的生物合成調(diào)控脂質(zhì)的沉積。ADAMTS4基因是牛肉形成大理石紋狀的重要基因之一。由于ADAMTS2和ADAMTS4基因都能對脂肪生成進行調(diào)控,從而推測ADAMTS2和ADAMTS4基因的表達與前體脂肪細胞分化和肌肉內(nèi)脂肪的沉積有關(guān)。因此,本文以牛前體脂肪細胞為研究對象,分析ADAMTS2或ADAMTS4基因?qū)χ|(zhì)沉積的影響,來獲得ADAMTS2和ADAMTS4基因在牛前體脂肪細胞分化過程中的作用。本研究首先采取犢牛皮下脂肪組織進行體外分離和培養(yǎng)。其次將合成的siRNA通過lipo-2000轉(zhuǎn)染至前體脂肪細胞中,特異性敲減ADAMTS2和ADAMTS4基因的表達,通過q RT-PCR方法檢測與脂肪細胞分化相關(guān)基因mRNA表達量變化情況,用胰島素等對轉(zhuǎn)染后的細胞進行誘導,通過HE染色、MASSON染色和掃描電鏡方法檢測脂質(zhì)沉積情況,Western-Blot來檢測轉(zhuǎn)染后蛋白表達情況。結(jié)果表明,敲減ADAMTS2基因后,與脂肪細胞分化相關(guān)基因PPAR、DGAT2、COL1A1、COL3A1、ADAMTS2、ADAMTS4和Aggrecan的mRNA表達量均降低,而與脂肪細胞分化相關(guān)基因EBP和COL2A1的mRNA表達量無明顯變化。敲減ADAMTS4基因后,與脂肪細胞分化相關(guān)基因PPAR、DGAT2、COL3A1、ADAMTS2、ADAMTS4和Aggrecan的mRNA表達量均降低,而與脂肪細胞分化相關(guān)基因COL1A1、EBP和COL2A1基因m RNA表達量無明顯變化。敲減ADAMTS2或ADAMTS4基因并經(jīng)過誘導后,實驗組脂質(zhì)量明顯比對照組少。Western-Blot分析表明,ADAMTS2和ADAMTS4蛋白在siRNA轉(zhuǎn)染后,蛋白表達量均降低。說明ADAMTS2和ADAMTS4基因均與脂肪細胞分化相關(guān),二者對前體脂肪細胞分化作用機制不同,本研究從形態(tài)學和分子調(diào)控機制方面對ADAMTS2和ADAMTS4基因在牛前體脂肪細胞分化中的作用進行了分析,這有助于揭示脂肪分化的分子機制,為提高牛肉品質(zhì)及良種選育提供理論依據(jù)。
[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.
【學位授予單位】：河南科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S823

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