法尼酯X受体对成纤维细胞生长因子21表达的影响及其机制的初步研究
发布时间：2019-03-25 10:44
【摘要】：研究背景:肥胖日益成为危害人体健康的重要因素,常伴有高胆固醇、高血压、高血脂及糖尿病等。成纤维细胞生长因子21(Fibroblast growth factor 21,FGF21)是新发现的代谢调控因子,大量实验研究证明,FGF21在糖脂代谢方面有着重要的调节功能,可以抵抗饮食诱导的肥胖,使肥胖动物体重显著降低。作为核受体超家族成员之一,法尼酯X受体(Farnesoid X receptor,FXR),可以通过调节诸多靶基因的表达而在调节糖代谢、脂代谢和胆固醇代谢中发挥着极其重要的作用,但FXR对FGF21表达的影响还未见报道。两者都高表达于肝脏,并在糖脂代谢中均发挥重要作用,我们推测FGF21是否也是FXR的一个靶基因呢?这将为了解FXR和FGF21的生物学功能及其机制,及防治肥胖提供重要的科学依据,并为之提供新的潜在治疗靶点。 研究目的:探讨FXR活化后是否能够调控FGF21的表达,并对其调控机制进行初步研究。 研究方法:1.本实验中分别给予为人肝癌细胞株HepG2和人胎肝细胞株L02 FXR不同浓度激动剂CDCA或GW4064处理24小时候,采用逆转录-PCR(RT-PCR)法、Realtime PCR法和Western Blot法分别检测FGF21mRNA和蛋白表达变化情况;2.在线预测FXR在FGF21基因启动子5’侧翼启动子区可能的结合位点,ER10分值最高,是FXR结合位点的可能性最大;提取HepG2细胞基因组DNA,并采用PCR法扩增包含ER10位点的FGF21基因启动子区,构建荧光素酶报告基因重组质粒pGL_3(-1790/+111),同时构建不包含ER10位点的pGL_3(-768/+111),利用脂质体,将活性FXR表达质粒Vp-FXR分别与两个重组质粒共转然到HepG2细胞中,24小时后检测各组报告基因的表达活性。3.随机将18只C57BL/6小鼠分为3组,根据小鼠的体重分别给予3组小鼠不同量的CDCA(0.1%DMSO,10mg/kg,50mg/kg)处理7天后处死,取其肝脏,采用RT-PCR法和Western-blot法分别检测小鼠肝脏中FGF21mRNA和蛋白的表达情况。 研究结果:1.FXR活化后可显著上调两种肝细胞中FGF21mRNA和蛋白表达水平,并呈剂量依赖型; 2.将荧光素酶报告基因重组载体pGL_3(-1790/+111)和pGL_3(-768/+111)分别与vp-FXR共转染入HepG2细胞后,pGL_3(-1790/+111)活性被显著上调,而pGL_3(-768/+111)活性无明显变化。3.CDCA可以显著上调C57BL/6小鼠肝脏中FGF21分子mRNA和蛋白的表达并呈剂量依赖型。 研究结论:以上实验结果证明,FXR活化后可在培养肝细胞和小鼠体内上调FGF21的表达,而FXR上调FGF21表达的分子机制可能是通过结合FGF21基因启动子上ER10序列来实现的,ER10可能是FXR的结合位点,而FXR对FGF21调控的具体机制还需进一步阐明。以上研究为阐明FXR和FGF21在肥胖中的作用及为有效防治肥胖提供重要的科学依据,并可能成为临床治疗肥胖的潜在重要靶点。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
【参考文献】
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1 董金瑜;刘红;彭家和;张艳;王强;李良鹏;王永超;江渝;;法尼酯X受体可下调肝细胞中肝型脂肪酸结合蛋白的表达[J];第三军医大学学报;2010年05期
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