山羊FGF1、FGF10和FGF21基因表达特性及其对山羊肌内前体脂肪细胞分化的影响

发布时间：2018-05-12 16:43

本文选题：山羊 + FGF1　；参考：《西南民族大学》2017年硕士论文

【摘要】：成纤维细胞生长因子家族(Fibroblast Growth Factors,FGFs)是一类多效性信号蛋白,参与动物的胚胎发育、代谢、神经功能、血管生成和肿瘤发生等过程。有研究表明FGF1、FGF10和FGF21在白色脂肪组织的发育、重塑和代谢中具有重要作用,但研究主要集中于小鼠和人,在山羊中尚未见相关报道,且NCBI上仅见山羊FGF1、FGF10和FGF21的预测序列,它们在山羊肌内前体脂肪细胞分化过程中是否具有调控作用及可能作用机制则需进行相关研究。本研究主要利用RT-PCR、实时荧光定量PCR(Real-time quantitative PCR,qPCR)、细胞培养、载体构建、超表达和干扰等方法阐明了FGF1、FGF10和FGF21基因的表达特性及对山羊肌内前体脂肪细胞分化的影响。获得主要研究结果如下:(1)克隆获得山羊FGF1、FGF10和FGF21基因序列分别为1 165 bp、1 252bp和666 bp,分别编码155、213和209个氨基酸。(2)FGF1、FGF10、FGF21及成纤维细胞生长因子受体(Fibroblast Growth Factor Receptors,FGFRs)在山羊各组织中广泛表达,FGF1在心脏和脂肪组织中高表达;FGF10在肺脏组织中表达水平最高;FGF21在肝脏和脂肪组织中高表达。FGFR1和FGFR2主要在肺脏组织中表达;FGFR3和FGFR4主要在肝脏组织中表达。(3)采用pAdEasy系统成功构建山羊重组腺病毒载体pAdEasy-FGF1,pAdEasy-FGF10和pAdEasy-FGF21,并在293A细胞中包装成功;针对FGF1、FGF10和FGF21基因的siRNA干扰效率分别达83.9%、83.5%和67.1%。(4)利用II型胶原酶与差速贴壁法分离获得了山羊原代肌内前体脂肪细胞;并明确了FGF1、FGF10、FGF21及FGFRs在山羊肌内前体脂肪细胞分化过程中的表达模式,即FGF1、FGF10和FGF21 mRNA表达水平均在诱导分化2 d后出现极显著升高(P0.01);FGFR1、FGFR2和FGFR3表达水平均在诱导分化8 d后达到最高(P0.01)。(5)超表达FGF1后脂肪细胞分化标志基因C/EBPα表达水平显著上调,PPARγ、AP2和SREBP1显著下调,FGFR1、FGFR2和FGFR4受体表达水平显著上调;超表达FGF10后脂肪细胞分化标志基因C/EBPα、LPL和四个受体FGFRs基因表达水平均显著上调,而AP2则显著下调;超表达FGF21后FGFR2和FGFR4两个受体表达水平分别显著上调,分化标志基因PPARγ、AP2和SREBP1表达水平分别显著下调。(6)干扰FGF1后脂肪细胞分化标志基因LPL显著下调,受体FGFR2显著上调而FGFR4则显著下调;干扰FGF10后,脂肪细胞分化标志基因LPL、C/EBPα、AP2、SREBP1及受体FGFR1、FGFR2、FGFR3、FGFR4均出现显著下调;干扰FGF21后,脂肪细胞分化标志基因PPARγ、C/EBPα和SREBP1显著上调,LPL显著下调,受体FGFR2和FGFR4也显著下调。以上结果表明FGF10和FGF21在山羊肌内脂肪细胞分化过程中可能分别具有促进和抑制作用,而FGF1具体起何种作用仍需进一步研究证明。本研究结果为进一步阐明FGF1、FGF10及FGF21基因调控山羊肌内脂肪细胞分化的分子机理提供了重要的数据支持。
[Abstract]:Fibroblast Growth FactorsFGFs (FGFs) are a class of multifunctional signaling proteins involved in embryonic development, metabolism, neural function, angiogenesis and tumorigenesis in animals. Some studies have shown that FGF1 FGF10 and FGF21 play an important role in the development, remodeling and metabolism of white adipose tissue. However, the studies mainly focus on mice and humans and have not been reported in goats, and only the predicted sequences of FGF1 FGF10 and FGF21 are found on NCBI. It is necessary to study whether they play a regulatory role in the differentiation of goat intramuscular precursor adipocytes and their possible mechanism. In this study, the expression characteristics of FGF1FGF10 and FGF21 genes and their effects on the differentiation of goat intramuscular precursor adipocytes were elucidated by RT-PCR, real-time quantitative PCR(Real-time quantitative PCR, cell culture, vector construction, overexpression and interference. The main results are as follows: 1: 1) the sequence of FGF1 FGF10 and FGF21 gene were 1 165bp1 252bp and 666bp, respectively, encoding 155213 and 209 amino acids, respectively. FGF1 FGF10 FGF21 and fibroblast Growth Factor ReceptorsFGFRswere widely used in goat tissues. High expression of FGF10 in heart and adipose tissue. FGF21 highly expressed in liver and adipose tissue. FGFR1 and FGFR2 were mainly expressed in lung tissue. FGFR3 and FGFR4 were mainly expressed in liver tissue. The recombinant adenovirus vectors pAdEasy-FGF1, pAdEasy-FGF10 and pAdEasy-FGF21 were successfully constructed by pAdEasy system and packaged successfully in 293A cells. The siRNA interference efficiency of FGF10 gene and FGF21 gene was 83.5% and 67.1%, respectively. Type II collagenase and differential adherent method were used to isolate the primary intramuscular adipocytes from goat. The expression patterns of FGF1, FGF10, FGF21 and FGFRs in the differentiation of goat intramuscular preadipocytes were determined. That is, the expression levels of FGF10 and FGF21 mRNA in FGF1 + FGF10 and FGF10 were significantly increased 2 days after induced differentiation. The expression levels of P0.01FGFR1FGFR2 and FGFR3 reached the highest level 8 days after differentiation) C/EBP 伪 expression of adipocyte differentiation marker gene C/EBP 伪 was upregulated significantly after FGF1 overexpression. PPAR- 纬 AP2 and SREBP1 down-regulated the expression of FGFR1FGFR2 and FGFR4 receptor. After overexpression of FGF10, the expression levels of C/EBP 伪 -LPL and four receptor FGFRs genes were significantly up-regulated, while AP2 was significantly down-regulated, FGFR2 and FGFR4 receptor expression levels were significantly up-regulated after overexpression of FGF21. The expression of differentiation marker gene PPAR 纬 -AP2 and SREBP1 were significantly down-regulated, respectively. After interfering with FGF1, adipocyte differentiation marker gene LPL was significantly down-regulated, receptor FGFR2 up-regulated and FGFR4 down-regulated, respectively, and after interfering with FGF10, adipocyte differentiation marker gene LPL was significantly down-regulated. The adipocyte differentiation marker gene, LPL-C / EBP 伪, AP2FGFRP1 and FGFR1 / FGFR2FGFR3FGFR4 were down-regulated, and the adipocyte differentiation marker genes PPAR 纬 -C / EBP 伪 and SREBP1 were up-regulated, and the receptors FGFR2 and FGFR4 were down-regulated. These results suggest that FGF10 and FGF21 may promote and inhibit the differentiation of goat intramuscular adipocytes respectively, but the specific role of FGF1 needs further study. The results provide important data for elucidating the molecular mechanism of FGF1 FGF10 and FGF21 gene regulating the differentiation of goat intramuscular adipocytes.
【学位授予单位】：西南民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

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