發(fā)布時(shí)間：2018-07-15 22:15

【摘要】：肌內(nèi)脂肪是影響豬肉風(fēng)味的重要因素之一,肌內(nèi)脂肪含量過(guò)低導(dǎo)致肉制品風(fēng)味下降。肌內(nèi)脂肪含量性狀與瘦肉率性狀之間呈負(fù)相關(guān),單靠遺傳育種同時(shí)解決豬的瘦肉率和肌內(nèi)脂肪含量問(wèn)題已經(jīng)遇到瓶頸。因此我們?cè)噲D尋求一種育種方式能在提高瘦肉率的同時(shí)又保證肌內(nèi)脂肪含量不降低。通過(guò)Hedgehog信號(hào)途徑在胚胎發(fā)育期調(diào)控間充質(zhì)干細(xì)胞向脂肪方向分化,以此機(jī)理指導(dǎo)豬的育種工作,篩選出提高豬的瘦肉率并能增加肌內(nèi)脂肪含量的最佳育種組合,為我國(guó)瘦肉型豬的培育和豬肉品質(zhì)調(diào)控研究提供新的研究思路。Hedgehog信號(hào)通路廣泛存在于脊椎動(dòng)物和無(wú)脊椎動(dòng)物體內(nèi),在動(dòng)物胚胎多種組織器官的生長(zhǎng)發(fā)育過(guò)程中發(fā)揮重要作用。近年來(lái),越來(lái)越多的國(guó)內(nèi)外研究已證明,Hedgehog信號(hào)通路在動(dòng)物脂肪細(xì)胞的生長(zhǎng)發(fā)育過(guò)程中發(fā)揮調(diào)控作用。激活動(dòng)物體內(nèi)Hedgehog信號(hào)通路能夠抑制脂肪細(xì)胞的生長(zhǎng)發(fā)育,然而反過(guò)來(lái)抑制動(dòng)物體內(nèi)的Hedgehog信號(hào)通路是否能促進(jìn)脂肪細(xì)胞的分化尚未明確。因此,本文就此問(wèn)題展開(kāi)研究,旨在尋找一種能在不降低瘦肉率的前提下提高肌內(nèi)脂肪含量的科學(xué)方法,為瘦肉型豬的培育和豬肉品質(zhì)調(diào)控研究提供參考依據(jù)。本研究旨在解析Hedgehog信號(hào)通路在小鼠胚胎間充質(zhì)干細(xì)胞成脂分化過(guò)程中發(fā)揮的作用,為瘦肉型豬的培育和豬肉品質(zhì)調(diào)控研究提供參考依據(jù)。本研究構(gòu)建小鼠間充質(zhì)干細(xì)胞C3H10T1/2細(xì)胞系為試驗(yàn)?zāi)Ｐ?用不同濃度環(huán)靶明(5μmol/L,10μmol/L,15μmol/L或20μmol/L)處理細(xì)胞,同時(shí)參考添加成脂誘導(dǎo)劑(10μg/mL胰島素,1mmol/L地塞米松,200μmol/L吲哚美辛,0.5mmol/L IBMX)對(duì)細(xì)胞的成脂分化誘導(dǎo)作用,分別在誘導(dǎo)的第1天、第2天、第3天、第7天、第14天和第21天提取細(xì)胞mRNA和蛋白用于后續(xù)試驗(yàn)。qRT-PCR法檢測(cè)Hedgehog信號(hào)通路相關(guān)基因、成脂分化調(diào)控基因PPARγ,C/EBPα以及成脂分化標(biāo)志基因FABP4在轉(zhuǎn)錄水平上的表達(dá)情況;采用western blot法檢測(cè)Hedgehog信號(hào)通路蛋白、成脂分化調(diào)控蛋白和FABP4蛋白的表達(dá)水平;利用油紅O染色法,檢測(cè)細(xì)胞內(nèi)脂滴形成情況。試驗(yàn)結(jié)果發(fā)現(xiàn),使用環(huán)靶明誘導(dǎo)C3H10T1/2細(xì)胞導(dǎo)致細(xì)胞Hedgehog信號(hào)通路各基因相對(duì)表達(dá)量下降,成脂分化調(diào)控基因以及成脂分化標(biāo)志基因FABP4相對(duì)表達(dá)量升高。其中10μmol/L環(huán)靶明處理的細(xì)胞效果最明顯,Hedgehog信號(hào)通路各基因相對(duì)表達(dá)量顯著下降(P0.05),成脂分化調(diào)控基因以及成脂分化標(biāo)志基因FABP4相對(duì)表達(dá)量升高(P0.05);使用10μmol/L環(huán)靶明誘導(dǎo)C3H10T1/2細(xì)胞,Hedgehog信號(hào)通路Shh蛋白和Gli1蛋白表達(dá)下降,PPARγ蛋白、C/EBPα蛋白和FABP4蛋白表達(dá)水平升高,同時(shí)油紅O染色方法證明細(xì)胞成脂分化作用增強(qiáng)。研究結(jié)果表明,環(huán)靶明能夠抑制Hedgehog信號(hào)通路,且最佳作用濃度為10μmol/L;環(huán)靶明通過(guò)抑制細(xì)胞Hedgehog信號(hào)通路對(duì)小鼠胚胎間充質(zhì)干細(xì)胞的成脂分化具有促進(jìn)作用。
[Abstract]:Intramuscular fat is one of the most important factors affecting pork flavor. There was a negative correlation between the traits of intramuscular fat content and lean meat rate. The problem of lean meat percentage and intramuscular fat content in pigs was solved simultaneously by genetic breeding. Therefore, we try to find a breeding method that can increase lean meat rate without decreasing intramuscular fat content. The differentiation of mesenchymal stem cells into fat was regulated by Hedgehog signaling pathway during embryonic development. The best breeding combination was selected to improve lean meat rate and increase intramuscular fat content. Hedgehog signaling pathway exists widely in vertebrates and invertebrates and plays an important role in the growth and development of various tissues and organs of animal embryos. In recent years, more and more studies at home and abroad have proved that Hedgehog signaling pathway plays a regulatory role in the growth and development of animal adipocytes. Activation of the Hedgehog signaling pathway can inhibit the growth and development of adipocytes, but it is not clear whether the Hedgehog signaling pathway can promote the differentiation of adipocytes. Therefore, the purpose of this study is to find a scientific method to improve the content of intramuscular fat without reducing lean meat rate, and to provide reference for the breeding of lean pigs and the regulation of pork quality. The purpose of this study was to elucidate the role of Hedgehog signaling pathway in adipogenic differentiation of mouse embryonic mesenchymal stem cells and to provide a reference for the breeding of lean pigs and the regulation of pork quality. In this study, mouse mesenchymal stem cells C3H10T1 / 2 cell lines were established as experimental models. The cells were treated with different concentrations of ring target (5 渭 mol / L ~ 10 渭 mol 路L ~ (-1) 15 渭 mol 路L ~ (-1 / L) or 20 渭 mol 路L ~ (-1) 路L ~ (-1). At the same time, the induction of adipogenic differentiation by adding 10 渭 g / mL insulin (10 渭 g / mL insulin 1 mmol / L dexamethasone 200 渭 mol / L indomethacin 0.5 mmol / L IBMX) on the 1st, 2nd, 3rd, 7th day of induction, On the 14th and 21st days, cell mRNA and protein were extracted to detect the expression of Hedgehog signaling pathway related genes, PPAR 緯 Cr / EBP 偽 and FABP4, respectively. The expression levels of Hedgehog signaling pathway protein, adipogenic differentiation regulatory protein and FABP4 protein were detected by western blot method, and lipid droplet formation was detected by oil red O staining. The results showed that the relative expression of genes in Hedgehog signaling pathway was decreased, the relative expression of adipogenic regulatory gene and FABP4, the marker gene of adipogenic differentiation, were increased in C3H10T1 / 2 cells induced by ring target. The relative expression of all genes in Hedgehog signaling pathway was significantly decreased (P0.05), the relative expression of adipogenic regulatory gene and FABP4 gene was increased (P0.05), and C3H10T1 / 2 was induced by 10 渭 mol / L ring target. The expression of Shh protein and Gli1 protein in Hedgehog signaling pathway decreased, and the expression levels of C / EBP 偽 protein and FABP4 protein increased in PPAR 緯 protein. At the same time, oil red O staining showed that the adipogenic differentiation of cells was enhanced. The results showed that the ring target could inhibit the Hedgehog signaling pathway with the best concentration of 10 渭 mol / L, and the ring target could promote the adipogenic differentiation of mouse embryonic mesenchymal stem cells by inhibiting the Hedgehog signaling pathway.
【學(xué)位授予單位】：浙江農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S828

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本文編號(hào)：2125549