本文選題：全反式維甲酸　切入點：骨髓間充質干細胞　出處：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：目的:研究激活蛋白(activator protein-1,AP-1)在全反式維甲酸(all-trans retinoic acid,ATRA)抑制大鼠骨髓間充質干細胞(bone marrow mesenchymal stem cell,BMSC)成脂分化信號通路中的調控機制。方法:采用SD大鼠原代BMSCs,體外分離,培養(yǎng)和成脂分化誘導。油紅O染色鑒定細胞成脂分化中脂滴形成情況。成脂誘導在第1天,第5天,第9天的同一時間點添加濃度為1μmol/L ATRA,持續(xù)處理24小時(即成脂分化誘導第2天,第6天,第10天),收取細胞樣本。應用熒光實時定量聚合酶鏈反應(real-time quantitative polymerase chain reaction,RT-PCR)檢測脂肪細胞形成相關基因脂肪酸結合蛋白(fatty acid-binding protein,FABP)、脂蛋白脂肪酶(lipoprotein lipase,LPL)、脂肪酸轉運蛋白(CD36)、脂滴包被蛋白(Perilipin)、過氧化物酶體增殖激活受體-γ2(peroxisome proliferator-activated receptor gamma-2,PPARγ2)以及AP-1家族各成員(Fosl1,Fosl2,c-Fos,Fos B,c-Jun,JunB,JunD)基因表達水平。Western blot檢測蛋白表達水平。染色質免疫共沉淀(chromatin immunoprecipitation,ChIP)檢測相關蛋白(RARγ,Fosl1)與PPARγ2基因是否存在相互作用。結果:(1)油紅O染色結果顯示,ATRA處理組中細胞形成的脂滴數量明顯減少,且油紅染料吸光度OD值明顯下降。(2)BMSCs成脂誘導12天后,RT-PCR結果顯示,與對照組相比,1μmol/L ATRA處理組脂肪細胞形成相關基因FABP,LPL,CD36,Perilipin,PPARγ2表達均顯著降低(PFABP0.05,PLPL0.001,PCD360.001,PPerilipin0.05),P0.05差異具有統計學意義。(3)RT-PCR檢測AP-1家族各轉錄因子表達,Fosl1在ATRA處理組成脂誘導第2天,第6天,第10天mRNA表達水平均顯著升高(P第2天0.01,P第6天0.01,P第10天0.001)。Westren blot結果顯示,ATRA處理組Fosl1蛋白表達顯著升高,而PPARγ2蛋白表達明顯下降。(4)ChIP-qPCR結果表明,Fosl1蛋白可結合在PPARγ2基因啟動子區(qū)域,而RARγ蛋白未直接結合在PPARγ2基因啟動子上。結論:ATRA可抑制BMSCs成脂分化及脂質代謝相關基因的表達,可能通過上調Fosl1直接結合PPARγ2基因啟動子區(qū)域下調其表達有關。
[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-緯 2(peroxisome proliferator-activated receptor gamma-2PPAR- 緯 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 緯 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 緯 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 緯 2 protein decreased significantly. The results of ChIP-qPCR showed that the Fosl1 protein could bind to the promoter region of PPAR 緯 2 gene. RAR 緯 protein was not directly bound to the promoter of PPAR 緯 2 gene. Conclusion RAR 緯 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 緯 2 gene by up-regulation of Fosl1.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R3416

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相關期刊論文 前1條

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本文編號：1654714