抑制Hedgehog信号通路对小鼠胚胎间充质干细胞成脂分化的影响
发布时间：2018-07-15 22:15
【摘要】：肌内脂肪是影响猪肉风味的重要因素之一,肌内脂肪含量过低导致肉制品风味下降。肌内脂肪含量性状与瘦肉率性状之间呈负相关,单靠遗传育种同时解决猪的瘦肉率和肌内脂肪含量问题已经遇到瓶颈。因此我们试图寻求一种育种方式能在提高瘦肉率的同时又保证肌内脂肪含量不降低。通过Hedgehog信号途径在胚胎发育期调控间充质干细胞向脂肪方向分化,以此机理指导猪的育种工作,筛选出提高猪的瘦肉率并能增加肌内脂肪含量的最佳育种组合,为我国瘦肉型猪的培育和猪肉品质调控研究提供新的研究思路。Hedgehog信号通路广泛存在于脊椎动物和无脊椎动物体内,在动物胚胎多种组织器官的生长发育过程中发挥重要作用。近年来,越来越多的国内外研究已证明,Hedgehog信号通路在动物脂肪细胞的生长发育过程中发挥调控作用。激活动物体内Hedgehog信号通路能够抑制脂肪细胞的生长发育,然而反过来抑制动物体内的Hedgehog信号通路是否能促进脂肪细胞的分化尚未明确。因此,本文就此问题展开研究,旨在寻找一种能在不降低瘦肉率的前提下提高肌内脂肪含量的科学方法,为瘦肉型猪的培育和猪肉品质调控研究提供参考依据。本研究旨在解析Hedgehog信号通路在小鼠胚胎间充质干细胞成脂分化过程中发挥的作用,为瘦肉型猪的培育和猪肉品质调控研究提供参考依据。本研究构建小鼠间充质干细胞C3H10T1/2细胞系为试验模型,用不同浓度环靶明(5μmol/L,10μmol/L,15μmol/L或20μmol/L)处理细胞,同时参考添加成脂诱导剂(10μg/mL胰岛素,1mmol/L地塞米松,200μmol/L吲哚美辛,0.5mmol/L IBMX)对细胞的成脂分化诱导作用,分别在诱导的第1天、第2天、第3天、第7天、第14天和第21天提取细胞mRNA和蛋白用于后续试验。qRT-PCR法检测Hedgehog信号通路相关基因、成脂分化调控基因PPARγ,C/EBPα以及成脂分化标志基因FABP4在转录水平上的表达情况;采用western blot法检测Hedgehog信号通路蛋白、成脂分化调控蛋白和FABP4蛋白的表达水平;利用油红O染色法,检测细胞内脂滴形成情况。试验结果发现,使用环靶明诱导C3H10T1/2细胞导致细胞Hedgehog信号通路各基因相对表达量下降,成脂分化调控基因以及成脂分化标志基因FABP4相对表达量升高。其中10μmol/L环靶明处理的细胞效果最明显,Hedgehog信号通路各基因相对表达量显著下降(P0.05),成脂分化调控基因以及成脂分化标志基因FABP4相对表达量升高(P0.05);使用10μmol/L环靶明诱导C3H10T1/2细胞,Hedgehog信号通路Shh蛋白和Gli1蛋白表达下降,PPARγ蛋白、C/EBPα蛋白和FABP4蛋白表达水平升高,同时油红O染色方法证明细胞成脂分化作用增强。研究结果表明,环靶明能够抑制Hedgehog信号通路,且最佳作用浓度为10μmol/L;环靶明通过抑制细胞Hedgehog信号通路对小鼠胚胎间充质干细胞的成脂分化具有促进作用。
[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 纬 Cr / EBP 伪 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 伪 protein and FABP4 protein increased in PPAR 纬 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.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
[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 纬 Cr / EBP 伪 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 伪 protein and FABP4 protein increased in PPAR 纬 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.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
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