【作者】 王乐；

【导师】 刘庆平；

【作者基本信息】 大连大学 ， 生物化学与分子生物学， 2014， 硕士

【摘要】 在抗动脉粥样硬化（atherosclerosis，AS）的研究中，维持细胞内胆固醇的平衡具有重要意义。胆固醇的逆向转运是肝脏外的胆固醇回归至肝脏，并最终以胆汁或排泄物的方式排出体外的过程，其中胆固醇的流出是一个重要部分，三磷酸腺苷结合盒转运体A1（ABCA1）的过表达可以促进胆固醇的流出，因此上调ABCA1的表达是抗动脉粥样硬化的关键因素之一。据报道，过氧化物酶体增殖物激活受体-γ（peroxisomeproliferator-activated receptor-γ，PPAR-γ）的激活调控可导致ABCA1的反式激活：当巨噬细胞吞噬氧化低密度脂蛋白（oxidized low density lipoprotein，ox-LDL）后，oxLDL的脂质成分释放出来，激活PPARγ的转录，一方面可促进ABCA1表达，促进胆固醇的流出，另一方面也刺激了清道夫受体CD36（scavenger receptor，CD36）的表达，导致细胞吞噬更多的oxLDL，从而维持机体内胆固醇的平衡。本实验室前期发现了oxLDL的脂质成分7-酮基胆甾醇-9-羧基壬烷（7-ketocholesteryl-9-carbo-xynonanoate，oxLig-1）与PPARγ的配体具有相似结构，那么oxLig-1是否参与调控PPARγ信号通路值得进一步研究。本实验将证实，在THP-1巨噬细胞中，oxLig-1经由PPARγ途径上调ABCA1的表达，促进胆固醇外流，同时也参与调控了PPARγ-CD36信号通路。为进一步研究oxLig-1对PPARγ介导的脂质代谢的提供依据和帮助。实验方法：应用ELISA方法检测oxLig-1与PPARγ配体结合区（ligand bindingdomain，LBD）的结合，双荧光素酶报告基因检测oxLig-1对PPARγ转录活性的影响，再用免疫印迹，3-Dodecanoyl-NBD-cholesterol (NBD-胆固醇)，流式细胞术，基因沉默等方法检测oxLig-1对由PPARγ介导的脂质代谢信号通路的影响。实验得出：oxLig-1在分子水平上可以与PPARγ-LBD结合，结合率高于PPARγ的合成配体曲格列酮（troglitazone，TRO），利用双荧光素酶报告基因检测表明oxLig-1提高了PPARγ的转录活性。在15-30μg/mL不同浓度中，oxLig-1均可上调ABCA1的表达，且20μg/mL，2h时ABCA1表达达到峰值，利用荧光标记胆固醇检测表明，在0-12h时oxLig-1可以促进胆固醇的流出，4h时外流率达到最高。在基因沉默实验中，PPARγ的沉默效率可达到90%左右，基因沉默后，ABCA1的表达和胆固醇的流出均受到不同程度的抑制，抑制率分别为62%和25%。而在oxLig-1作用细胞24h时，CD36的表达也有所上调，且受到PPARγ的调控。结论：oxLig-1可能是PPARγ的新型配体，并经由此通路上调ABCA1和CD36的表达，在维持体内胆固醇的平衡中发挥重要作用。更多还原

【Abstract】 It has a positive impact that removed excess cholesterol from the intracellular to the liverin atherosclerosis.Reverse cholesterol transport (RCT) is a pathway by which to describe theProcess by which extrahepatic (peripheral) cholesterol is returned to the liver for excretion inthe bile and ultimately the feces.Cholesterol efflux,part of the RCT process, is a major processby which macrophages within the vessel wall secrete cholesterol outside cells.Overexpressionof ATP binding cassette transporter A1(ABCA1) stimulates efflux of cholesterol,ABCA1istherefore an important target for anti-atherogenic therapies. It has been reported that activationof Peroxisome proliferator-activated receptor γ(PPARγ) by natural or synthetic ligands resultsin thetransactivation of ABCA1:Initially, uptake of oxidized lowdensity lipoprotein(oxLDL)by macrophages results in induction of PPARγ,release of its ligands, on the one hand，increasedexpression of ABCA1，promoted thecholesterol efflux,on theother hand,up-regulated expression of scavenger receptor CD36and stimulate uptake of oxLDL,keepcholesterol balance.We previously purified a negatively charged lipid moiety from Cu2+induced oxLDL,7-ketocholesteryl-9-carboxynonanoate(oxLig-1),which have the similarstructure with PPARγ ligands.Theeffect of oxLig-1onABCA1expressionmediated by nuclearreceptors (NRs) has not been elucidated.In the present study, we investigated the signalprocess by which oxLig-1regulates ABCA1expression and promotes ApoA-I-mediatedcholesterol efflux from macrophages by the mediation of PPARγ.Methods: First,we docked oxLig-1and the ligand-binding domain (LBD) of PPARγ byELISA assay.Dual-Luciferase reporter assay detected the effect of oxLig-1on PPARγtranscriptional activity.Then analysed the influence of oxLig-1on PPARγ-med iated Lipidmetabolic signaling pathways by the methods of Western blotting,3-Dodecanoyl-NBD-chol-esterol(NBD-Cholesterol),Flow cytometry and shRNA.Results: It was found that oxLig-1was able to bind to PPARγ LBD,and oxLig-1was aneffective competitor for troglitazone binding.oxLig-1also improve the transcription activityof PPARγ.ABCA1expression was increased in the presence of oxLig-1at differentconcentrations (15~30μg/mL) within6h. The expression of ABCA1reached the peak levelwith20μg/mL oxLig-1for2hrs. A peak efflux rate was observed with20μg/mL oxLig-1for4h.As expected, PPARγ-shRNA resulted in a reduction of90%in PPARγ proteinexpression.Under PPARγ knockdown, oxLig-1-induced ABCA1expression and cholesterolefflux was blocked by62%and25%respectively. CD36expression was increased in thepresence of oxLig-1also mediated by PPARγ.Conclusions:These observations suggest that oxLig-1is possibly a novel ligand for PPARγ, play an important role in regulating lipid metabolism balance by increasingABCA1,CD36expression via induction of PPARγ.更多还原