全反式维甲酸抑制大鼠骨髓间充质干细胞成脂分化信号通路中Fosl1直接调控PPARγ2

发布时间：2018-03-23 18:44

本文选题：全反式维甲酸　切入点：骨髓间充质干细胞　出处：《重庆医科大学》2017年硕士论文

【摘要】：目的:研究激活蛋白(activator protein-1,AP-1)在全反式维甲酸(all-trans retinoic acid,ATRA)抑制大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)成脂分化信号通路中的调控机制。方法:采用SD大鼠原代BMSCs,体外分离,培养和成脂分化诱导。油红O染色鉴定细胞成脂分化中脂滴形成情况。成脂诱导在第1天,第5天,第9天的同一时间点添加浓度为1μmol/L ATRA,持续处理24小时(即成脂分化诱导第2天,第6天,第10天),收取细胞样本。应用荧光实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-PCR)检测脂肪细胞形成相关基因脂肪酸结合蛋白(fatty acid-binding protein,FABP)、脂蛋白脂肪酶(lipoprotein lipase,LPL)、脂肪酸转运蛋白(CD36)、脂滴包被蛋白(Perilipin)、过氧化物酶体增殖激活受体-γ2(peroxisome proliferator-activated receptor gamma-2,PPARγ2)以及AP-1家族各成员(Fosl1,Fosl2,c-Fos,Fos B,c-Jun,JunB,JunD)基因表达水平。Western blot检测蛋白表达水平。染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)检测相关蛋白(RARγ,Fosl1)与PPARγ2基因是否存在相互作用。结果:(1)油红O染色结果显示,ATRA处理组中细胞形成的脂滴数量明显减少,且油红染料吸光度OD值明显下降。(2)BMSCs成脂诱导12天后,RT-PCR结果显示,与对照组相比,1μmol/L ATRA处理组脂肪细胞形成相关基因FABP,LPL,CD36,Perilipin,PPARγ2表达均显著降低(PFABP0.05,PLPL0.001,PCD360.001,PPerilipin0.05),P0.05差异具有统计学意义。(3)RT-PCR检测AP-1家族各转录因子表达,Fosl1在ATRA处理组成脂诱导第2天,第6天,第10天mRNA表达水平均显著升高(P第2天0.01,P第6天0.01,P第10天0.001)。Westren blot结果显示,ATRA处理组Fosl1蛋白表达显著升高,而PPARγ2蛋白表达明显下降。(4)ChIP-qPCR结果表明,Fosl1蛋白可结合在PPARγ2基因启动子区域,而RARγ蛋白未直接结合在PPARγ2基因启动子上。结论:ATRA可抑制BMSCs成脂分化及脂质代谢相关基因的表达,可能通过上调Fosl1直接结合PPARγ2基因启动子区域下调其表达有关。
[Abstract]:Aim: to study the regulatory mechanism of activator protein-1 (AP-1) in inhibiting adipogenic differentiation signaling pathway of rat bone marrow mesenchymal stem cells (BMSCs) by all-trans retinoic ATRA.Methods: SD rat primary BMSCs were isolated in vitro. Oil red O staining was used to identify the formation of lipid droplets in adipogenic differentiation. At the same time point on the 9th day, 1 渭 mol/L ATRA was added for 24 hours (that is, the second day, the sixth day of adipogenic induction, the second day and the sixth day of adipogenic induction). On day 10, cell samples were collected. Real-time quantitative polymerase chain reactionation assay (RT-PCRs) was used to detect fatty acid binding protein (acid-binding), lipoprotein lipase lipase, fatty acid transporter CD36 and lipid droplets in adipocytes. Perilipinus, peroxisome proliferation-activated receptor-纬 2(peroxisome proliferator-activated receptor gamma-2PPAR- 纬 2) and members of the AP-1 family, Fosl1c Fosl2Fosl2Fosl2Fos Bc-JunBJunD. Western blot was used to detect protein expression. Chromatin immunoprecipitation assay (Chromatin) and PPAR 纬 2 gene were detected by chromatin immunoprecipitation assay. Results the oil red O staining showed that the number of lipid droplets formed in the cells of the ATRA treatment group was significantly reduced. The OD value of oil red dye decreased significantly. Compared with the control group, the expression of the adipocyte formation related gene FABP- LPL36, CD36, PPAR 纬 2 in the 1 渭 mol/L ATRA treatment group was significantly lower than that in the control group. The expression of Fosl1 in the AP-1 family was detected by RT-PCR on the 2nd and 6th day of lipid induction by ATRA treatment, and the expression of Fosl1 was detected on the 2nd and 6th day after the ATRA treatment, and the expression of Fosl1 in the AP-1 family was detected on the 2nd and 6th day after the ATRA treatment, and the expression of Fosl1 in the AP-1 family was detected by reverse transcription-polymerase chain reaction (RT-PCR). On the 10th day, the expression level of mRNA was significantly increased. The results of 0.001).Westren blot on the 2nd day, the 2nd day, the 6th day, the 6th day, the 0.001).Westren blot showed that the expression of Fosl1 protein increased significantly, while the expression of PPAR 纬 2 protein decreased significantly. The results of ChIP-qPCR showed that the Fosl1 protein could bind to the promoter region of PPAR 纬 2 gene. RAR 纬 protein was not directly bound to the promoter of PPAR 纬 2 gene. Conclusion RAR 纬 protein can inhibit the expression of BMSCs adipogenic differentiation and lipid metabolism related genes, which may be related to the down-regulation of the promoter region of PPAR 纬 2 gene by up-regulation of Fosl1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3416

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