本文選題：山羊 + FGF1　； 參考：《西南民族大學(xué)》2017年碩士論文

【摘要】：成纖維細(xì)胞生長(zhǎng)因子家族(Fibroblast Growth Factors,FGFs)是一類多效性信號(hào)蛋白,參與動(dòng)物的胚胎發(fā)育、代謝、神經(jīng)功能、血管生成和腫瘤發(fā)生等過程。有研究表明FGF1、FGF10和FGF21在白色脂肪組織的發(fā)育、重塑和代謝中具有重要作用,但研究主要集中于小鼠和人,在山羊中尚未見相關(guān)報(bào)道,且NCBI上僅見山羊FGF1、FGF10和FGF21的預(yù)測(cè)序列,它們?cè)谏窖蚣?nèi)前體脂肪細(xì)胞分化過程中是否具有調(diào)控作用及可能作用機(jī)制則需進(jìn)行相關(guān)研究。本研究主要利用RT-PCR、實(shí)時(shí)熒光定量PCR(Real-time quantitative PCR,qPCR)、細(xì)胞培養(yǎng)、載體構(gòu)建、超表達(dá)和干擾等方法闡明了FGF1、FGF10和FGF21基因的表達(dá)特性及對(duì)山羊肌內(nèi)前體脂肪細(xì)胞分化的影響。獲得主要研究結(jié)果如下:(1)克隆獲得山羊FGF1、FGF10和FGF21基因序列分別為1 165 bp、1 252bp和666 bp,分別編碼155、213和209個(gè)氨基酸。(2)FGF1、FGF10、FGF21及成纖維細(xì)胞生長(zhǎng)因子受體(Fibroblast Growth Factor Receptors,FGFRs)在山羊各組織中廣泛表達(dá),FGF1在心臟和脂肪組織中高表達(dá);FGF10在肺臟組織中表達(dá)水平最高;FGF21在肝臟和脂肪組織中高表達(dá)。FGFR1和FGFR2主要在肺臟組織中表達(dá);FGFR3和FGFR4主要在肝臟組織中表達(dá)。(3)采用pAdEasy系統(tǒng)成功構(gòu)建山羊重組腺病毒載體pAdEasy-FGF1,pAdEasy-FGF10和pAdEasy-FGF21,并在293A細(xì)胞中包裝成功;針對(duì)FGF1、FGF10和FGF21基因的siRNA干擾效率分別達(dá)83.9%、83.5%和67.1%。(4)利用II型膠原酶與差速貼壁法分離獲得了山羊原代肌內(nèi)前體脂肪細(xì)胞;并明確了FGF1、FGF10、FGF21及FGFRs在山羊肌內(nèi)前體脂肪細(xì)胞分化過程中的表達(dá)模式,即FGF1、FGF10和FGF21 mRNA表達(dá)水平均在誘導(dǎo)分化2 d后出現(xiàn)極顯著升高(P0.01);FGFR1、FGFR2和FGFR3表達(dá)水平均在誘導(dǎo)分化8 d后達(dá)到最高(P0.01)。(5)超表達(dá)FGF1后脂肪細(xì)胞分化標(biāo)志基因C/EBPα表達(dá)水平顯著上調(diào),PPARγ、AP2和SREBP1顯著下調(diào),FGFR1、FGFR2和FGFR4受體表達(dá)水平顯著上調(diào);超表達(dá)FGF10后脂肪細(xì)胞分化標(biāo)志基因C/EBPα、LPL和四個(gè)受體FGFRs基因表達(dá)水平均顯著上調(diào),而AP2則顯著下調(diào);超表達(dá)FGF21后FGFR2和FGFR4兩個(gè)受體表達(dá)水平分別顯著上調(diào),分化標(biāo)志基因PPARγ、AP2和SREBP1表達(dá)水平分別顯著下調(diào)。(6)干擾FGF1后脂肪細(xì)胞分化標(biāo)志基因LPL顯著下調(diào),受體FGFR2顯著上調(diào)而FGFR4則顯著下調(diào);干擾FGF10后,脂肪細(xì)胞分化標(biāo)志基因LPL、C/EBPα、AP2、SREBP1及受體FGFR1、FGFR2、FGFR3、FGFR4均出現(xiàn)顯著下調(diào);干擾FGF21后,脂肪細(xì)胞分化標(biāo)志基因PPARγ、C/EBPα和SREBP1顯著上調(diào),LPL顯著下調(diào),受體FGFR2和FGFR4也顯著下調(diào)。以上結(jié)果表明FGF10和FGF21在山羊肌內(nèi)脂肪細(xì)胞分化過程中可能分別具有促進(jìn)和抑制作用,而FGF1具體起何種作用仍需進(jìn)一步研究證明。本研究結(jié)果為進(jìn)一步闡明FGF1、FGF10及FGF21基因調(diào)控山羊肌內(nèi)脂肪細(xì)胞分化的分子機(jī)理提供了重要的數(shù)據(jù)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 偽 expression of adipocyte differentiation marker gene C/EBP 偽 was upregulated significantly after FGF1 overexpression. PPAR- 緯 AP2 and SREBP1 down-regulated the expression of FGFR1FGFR2 and FGFR4 receptor. After overexpression of FGF10, the expression levels of C/EBP 偽 -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 緯 -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 偽, AP2FGFRP1 and FGFR1 / FGFR2FGFR3FGFR4 were down-regulated, and the adipocyte differentiation marker genes PPAR 緯 -C / EBP 偽 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.
【學(xué)位授予單位】：西南民族大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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