法尼酯X受體對(duì)成纖維細(xì)胞生長(zhǎng)因子21表達(dá)的影響及其機(jī)制的初步研究
發(fā)布時(shí)間：2019-03-25 10:44
【摘要】：研究背景:肥胖日益成為危害人體健康的重要因素,常伴有高膽固醇、高血壓、高血脂及糖尿病等。成纖維細(xì)胞生長(zhǎng)因子21(Fibroblast growth factor 21,FGF21)是新發(fā)現(xiàn)的代謝調(diào)控因子,大量實(shí)驗(yàn)研究證明,FGF21在糖脂代謝方面有著重要的調(diào)節(jié)功能,可以抵抗飲食誘導(dǎo)的肥胖,使肥胖動(dòng)物體重顯著降低。作為核受體超家族成員之一,法尼酯X受體(Farnesoid X receptor,FXR),可以通過調(diào)節(jié)諸多靶基因的表達(dá)而在調(diào)節(jié)糖代謝、脂代謝和膽固醇代謝中發(fā)揮著極其重要的作用,但FXR對(duì)FGF21表達(dá)的影響還未見報(bào)道。兩者都高表達(dá)于肝臟,并在糖脂代謝中均發(fā)揮重要作用,我們推測(cè)FGF21是否也是FXR的一個(gè)靶基因呢?這將為了解FXR和FGF21的生物學(xué)功能及其機(jī)制,及防治肥胖提供重要的科學(xué)依據(jù),并為之提供新的潛在治療靶點(diǎn)。 研究目的:探討FXR活化后是否能夠調(diào)控FGF21的表達(dá),并對(duì)其調(diào)控機(jī)制進(jìn)行初步研究。 研究方法:1.本實(shí)驗(yàn)中分別給予為人肝癌細(xì)胞株HepG2和人胎肝細(xì)胞株L02 FXR不同濃度激動(dòng)劑CDCA或GW4064處理24小時(shí)候,采用逆轉(zhuǎn)錄-PCR(RT-PCR)法、Realtime PCR法和Western Blot法分別檢測(cè)FGF21mRNA和蛋白表達(dá)變化情況;2.在線預(yù)測(cè)FXR在FGF21基因啟動(dòng)子5’側(cè)翼啟動(dòng)子區(qū)可能的結(jié)合位點(diǎn),ER10分值最高,是FXR結(jié)合位點(diǎn)的可能性最大;提取HepG2細(xì)胞基因組DNA,并采用PCR法擴(kuò)增包含ER10位點(diǎn)的FGF21基因啟動(dòng)子區(qū),構(gòu)建熒光素酶報(bào)告基因重組質(zhì)粒pGL_3(-1790/+111),同時(shí)構(gòu)建不包含ER10位點(diǎn)的pGL_3(-768/+111),利用脂質(zhì)體,將活性FXR表達(dá)質(zhì)粒Vp-FXR分別與兩個(gè)重組質(zhì)粒共轉(zhuǎn)然到HepG2細(xì)胞中,24小時(shí)后檢測(cè)各組報(bào)告基因的表達(dá)活性。3.隨機(jī)將18只C57BL/6小鼠分為3組,根據(jù)小鼠的體重分別給予3組小鼠不同量的CDCA(0.1%DMSO,10mg/kg,50mg/kg)處理7天后處死,取其肝臟,采用RT-PCR法和Western-blot法分別檢測(cè)小鼠肝臟中FGF21mRNA和蛋白的表達(dá)情況。 研究結(jié)果:1.FXR活化后可顯著上調(diào)兩種肝細(xì)胞中FGF21mRNA和蛋白表達(dá)水平,并呈劑量依賴型; 2.將熒光素酶報(bào)告基因重組載體pGL_3(-1790/+111)和pGL_3(-768/+111)分別與vp-FXR共轉(zhuǎn)染入HepG2細(xì)胞后,pGL_3(-1790/+111)活性被顯著上調(diào),而pGL_3(-768/+111)活性無(wú)明顯變化。3.CDCA可以顯著上調(diào)C57BL/6小鼠肝臟中FGF21分子mRNA和蛋白的表達(dá)并呈劑量依賴型。 研究結(jié)論:以上實(shí)驗(yàn)結(jié)果證明,FXR活化后可在培養(yǎng)肝細(xì)胞和小鼠體內(nèi)上調(diào)FGF21的表達(dá),而FXR上調(diào)FGF21表達(dá)的分子機(jī)制可能是通過結(jié)合FGF21基因啟動(dòng)子上ER10序列來實(shí)現(xiàn)的,ER10可能是FXR的結(jié)合位點(diǎn),而FXR對(duì)FGF21調(diào)控的具體機(jī)制還需進(jìn)一步闡明。以上研究為闡明FXR和FGF21在肥胖中的作用及為有效防治肥胖提供重要的科學(xué)依據(jù),并可能成為臨床治療肥胖的潛在重要靶點(diǎn)。
[Abstract]:Background: obesity is increasingly becoming an important factor that endangers human health, often accompanied by high cholesterol, hypertension, hyperlipidemia and diabetes. Fibroblast growth factor 21 (Fibroblast growth factor 21 (FGF21) is a newly discovered metabolic regulatory factor. A large number of experimental studies have shown that FGF21 plays an important role in glucose and lipid metabolism and can resist diet-induced obesity. Reduce the weight of obese animals significantly. As a member of the nuclear receptor superfamily, Farni X receptor (Farnesoid X receptor,FXR plays an important role in the regulation of glucose metabolism, lipid metabolism and cholesterol metabolism by regulating the expression of many target genes. However, the effect of FXR on the expression of FGF21 has not been reported. Both of them are highly expressed in the liver and play an important role in glucose and lipid metabolism. We speculate whether FGF21 is also a target gene of FXR. This will provide an important scientific basis for understanding the biological function and mechanism of FXR and FGF21, as well as the prevention and treatment of obesity, and provide a new potential therapeutic target for it. Objective: to investigate whether the activation of FXR can regulate the expression of FGF21 and the mechanism of its regulation. Methods of research: 1. Human hepatoma cell line HepG2 and human fetal liver cell line L02 FXR were treated with CDCA or GW4064 at different concentrations for 24 hours. Reverse transcription-PCR (RT-PCR) method was used. The expression of FGF21mRNA and protein were detected by Realtime PCR and Western Blot, respectively. 2. On-line prediction of the possible binding sites of FXR in the 5 'flanking promoter region of FGF21 gene showed that ER10 score was the highest and FXR binding site was the most likely. Genomic DNA, was extracted from HepG2 cells and the promoter region of FGF21 gene containing ER10 site was amplified by PCR. Luciferase reporter gene recombinant plasmid pGL_3 (- 1790 / 111) was constructed, and pGL_3 (- 768 / 111) without ER10 site was constructed. Using liposomes, the active FXR expression plasmid Vp-FXR was co-transfected with two recombinant plasmids into HepG2 cells. 24 hours later, the expression activity of reporter genes in each group was detected. 3. Eighteen C57BL/6 mice were randomly divided into three groups. According to their weight, the mice in the three groups were treated with different doses of CDCA (0.1% DMSO, 10 mg / kg, 50 mg / kg) for 7 days, and their livers were taken out. The expression of FGF21mRNA and protein in mouse liver were detected by RT- PCR and Western-blot, respectively. The results showed that 1.FXR could significantly up-regulate the expression of FGF21mRNA and protein in two kinds of hepatocytes in a dose-dependent manner. When luciferase reporter gene recombinant vectors pGL_3 (- 1790 / 111) and pGL_3 (- 768 / 111) were co-transfected into HepG2 cells with vp-FXR, the activity of pGL_3 (- 1790 / 111) was up-regulated significantly. PGL_3 (- 768 / 111) activity did not change significantly. 3. CDCA could significantly up-regulate the expression of FGF21 mRNA and protein in the liver of C57BL/6 mice in a dose-dependent manner. Conclusion: these results suggest that FXR can up-regulate the expression of FGF21 in cultured hepatocytes and mice, and the molecular mechanism of up-regulation of FGF21 expression by FXR may be achieved by binding the ER10 sequence on the promoter of FGF21 gene. ER10 may be the binding site of FXR, and the specific mechanism of FXR regulating FGF21 needs further elucidation. These studies provide important scientific basis for elucidating the role of FXR and FGF21 in obesity and providing important scientific basis for effective prevention and treatment of obesity, and may become potential important targets for clinical treatment of obesity.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
本文編號(hào)：2446898
[Abstract]:Background: obesity is increasingly becoming an important factor that endangers human health, often accompanied by high cholesterol, hypertension, hyperlipidemia and diabetes. Fibroblast growth factor 21 (Fibroblast growth factor 21 (FGF21) is a newly discovered metabolic regulatory factor. A large number of experimental studies have shown that FGF21 plays an important role in glucose and lipid metabolism and can resist diet-induced obesity. Reduce the weight of obese animals significantly. As a member of the nuclear receptor superfamily, Farni X receptor (Farnesoid X receptor,FXR plays an important role in the regulation of glucose metabolism, lipid metabolism and cholesterol metabolism by regulating the expression of many target genes. However, the effect of FXR on the expression of FGF21 has not been reported. Both of them are highly expressed in the liver and play an important role in glucose and lipid metabolism. We speculate whether FGF21 is also a target gene of FXR. This will provide an important scientific basis for understanding the biological function and mechanism of FXR and FGF21, as well as the prevention and treatment of obesity, and provide a new potential therapeutic target for it. Objective: to investigate whether the activation of FXR can regulate the expression of FGF21 and the mechanism of its regulation. Methods of research: 1. Human hepatoma cell line HepG2 and human fetal liver cell line L02 FXR were treated with CDCA or GW4064 at different concentrations for 24 hours. Reverse transcription-PCR (RT-PCR) method was used. The expression of FGF21mRNA and protein were detected by Realtime PCR and Western Blot, respectively. 2. On-line prediction of the possible binding sites of FXR in the 5 'flanking promoter region of FGF21 gene showed that ER10 score was the highest and FXR binding site was the most likely. Genomic DNA, was extracted from HepG2 cells and the promoter region of FGF21 gene containing ER10 site was amplified by PCR. Luciferase reporter gene recombinant plasmid pGL_3 (- 1790 / 111) was constructed, and pGL_3 (- 768 / 111) without ER10 site was constructed. Using liposomes, the active FXR expression plasmid Vp-FXR was co-transfected with two recombinant plasmids into HepG2 cells. 24 hours later, the expression activity of reporter genes in each group was detected. 3. Eighteen C57BL/6 mice were randomly divided into three groups. According to their weight, the mice in the three groups were treated with different doses of CDCA (0.1% DMSO, 10 mg / kg, 50 mg / kg) for 7 days, and their livers were taken out. The expression of FGF21mRNA and protein in mouse liver were detected by RT- PCR and Western-blot, respectively. The results showed that 1.FXR could significantly up-regulate the expression of FGF21mRNA and protein in two kinds of hepatocytes in a dose-dependent manner. When luciferase reporter gene recombinant vectors pGL_3 (- 1790 / 111) and pGL_3 (- 768 / 111) were co-transfected into HepG2 cells with vp-FXR, the activity of pGL_3 (- 1790 / 111) was up-regulated significantly. PGL_3 (- 768 / 111) activity did not change significantly. 3. CDCA could significantly up-regulate the expression of FGF21 mRNA and protein in the liver of C57BL/6 mice in a dose-dependent manner. Conclusion: these results suggest that FXR can up-regulate the expression of FGF21 in cultured hepatocytes and mice, and the molecular mechanism of up-regulation of FGF21 expression by FXR may be achieved by binding the ER10 sequence on the promoter of FGF21 gene. ER10 may be the binding site of FXR, and the specific mechanism of FXR regulating FGF21 needs further elucidation. These studies provide important scientific basis for elucidating the role of FXR and FGF21 in obesity and providing important scientific basis for effective prevention and treatment of obesity, and may become potential important targets for clinical treatment of obesity.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 董金瑜;劉紅;彭家和;張艷;王強(qiáng);李良鵬;王永超;江渝;;法尼酯X受體可下調(diào)肝細(xì)胞中肝型脂肪酸結(jié)合蛋白的表達(dá)[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2010年05期
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