【作者】 陈江源；

【导师】 屈伸；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 油酸是食用油的主要成分。已有研究表明，在肠粘膜上皮细胞中，与其它脂肪酸相比，油酸是一种公认的具有强的诱导富含甘油三酯脂蛋白合成与分泌的脂肪酸，它通过促进胞膜胆固醇向内质网运输，增加胆固醇及其酯的分泌，此外，在无外源胆固醇的条件下，油酸可促进胆固醇的合成。动物实验结果表明，在高胆固醇的条件下，高浓度的油酸可降低胆固醇的吸收率，流行病学调查的结果表明，油酸具有降低血浆LDL胆固醇浓度的作用，与多不饱和脂酸一道被称为“健康脂酸”。然而，在肠粘膜上皮细胞中，油酸影响胆固醇的吸收与运输机制，目前还不清楚。高胆固醇血症是导致动脉粥样硬化的重要危险因子。除机体自身合成的胆固醇外，食物胆固醇是导致高胆固醇血症的重要因素，减少小肠对食物与胆汁胆固醇的吸收有利于降低血液胆固醇的水平；因此小肠在保持机体胆固醇稳态方面发挥重要作用。NPC1L1(Niemann-Pick C1-Like 1)，分布在肠粘膜上皮细胞刷状缘侧，是负责胆固醇吸收的重要转运体，NPC1L1敲除的小鼠完全抵抗食物诱导的高胆固醇血症，即使用高胆固醇的食物饲养，也不会患高胆固醇血症。ABCG5／8异二聚体也分布在肠粘膜上皮细胞刷状缘侧，通过促进固醇的外排调节胆固醇的吸收。HMGCR是胆固醇合成的限速酶。部分合成与吸收的胆固醇在内质网上经酰基辅酶A：胆固醇酰基转移酶2(Acy1-Coenzyme A CholesterolAcyltransferase，ACAT2)催化形成胆固醇酯。在肠与肝中，富含甘油三酯的酯蛋白的装配与分泌，MTP(Microsomal triglyceride transfer protein)是必不可少的重要转运体。固醇调节元件结合蛋白-2(sterol regulatoryelement-binding protein-2，SREBP-2)是结合在内质网上的转录因子，经蛋白裂解活化，促进胆固醇合成与吸收基因的表达。已有文献报道，它正向调节NPC1L1、HMGCR(3-hydroxy-3-methylglutaryl-CoA reductase)的表达，负向调节MTP的表达。内质网是蛋白质合成、折叠，合成胆固醇及脂酸，维持钙离子稳态的重要场所。当内质网的功能受到干扰，内质网应激发生，通过内质网应激活化一系列基因的表达，增加内质网处理未折叠蛋白的能力，减少内质网应激造成的压力。已有文献报道，内质网应激通过未折叠蛋白反应(unfolded protein response，UPR)信号通路促进或抑制SREBP-2的成熟，进而调节其调控的基因的表达。近年来的研究表明，内质网应激通过未折叠蛋白反应(UPR)的不同信号通路正向或负向调节SREBP-2调控的基因表达。已有文献报道，油酸可引起肝细胞发生内质网应激，从而影响apoB100的分泌。但是，在肠粘膜上皮细胞胆固醇吸收过程中，油酸是否可引起内质网应激，进而影响胆固醇转运相关蛋白的表达，目前还不是很清楚．本文用肠粘膜上皮细胞CaCo-2作模型，用各种脂质微团温育细胞，探讨油酸对肠粘膜上皮细胞胆固醇吸收、转运相关蛋白表达的影响，以及油酸诱导的UPR对胆固醇关键转运蛋白的调节作用．用牛磺胆酸钠(TC 5mM)，TC与胆固醇(CHOL 0．2mM)、TC与油酸(0．25mM，0．5mM，1．0 mM)及胆固醇(CHOL 0．2mM)、以及TC与油酸(0．25mM，0．5mM，1．0 mM)制备的脂质微团孵育CaCo-2细胞，24小时后，用RT-PCR检测NPC1L1、ABCG8、HMGCR、ACAT-2、MTP、剪切型XBP1、CHOP mRNA水平的变化，用Western Blotting检测BIP、ATF6(Activating transcription factor-6)、SREBP2、NPC1L1、ABCG8蛋白水平的变化。并利用UPR的抑制剂4-苯基丁酸(4-phenyl-butyric acid，PBA)抑制UPR，阐明UPR在调节胆固醇相关转运蛋白表达中的作用。实验结果显示：用不同的脂质微团孵育CaCo-2细胞，在mRNA与蛋白水平ACAT-2的表达无变化；蛋白水平SREBP-2的表达无变化；在有或无微团胆固醇的条件下，高浓度的油酸(0．5mM、1mM)增加了剪切型XBP1(X-box bindingprotein 1)mRNA的数量，增加了BIP(immunoglobulin heavy chain-bindingprotein)蛋白及CHOP(DNA-damage-inducible transcript 3)的表达，促进了ATF-6的成熟，诱发了内质网应激；在有微团胆固醇的条件下，油酸以浓度依赖的方式增加了ABCG8的表达；降低了NPC1L1、HMGCR的表达，MTP的表达显著增加；相反，在无微团胆固醇存在的条件下，油酸增加了NPC1L1、HMGCR的表达。用UPR的抑制剂PBA处理细胞，PBA抑制了XBP1的选择性剪接与ATF-6的成熟，消除了油酸引起的CHOP和BIP表达上调，抑制了内质网应激的发生。用PBA处理细胞，抑制了高浓度的油酸通过内质网应激引起的NPC1L1、HMGCR、MTP的表达变化。然而，PBA对ABCG8的表达无影响。总之，在有或无外源胆固醇的条件下，高浓度的油酸通过未折叠蛋白反应差异地调节肠粘膜上皮细胞NPC1L1、HMGCR、MTP的表达。在有大量外源胆固醇的条件下，高浓度的油酸通过折叠蛋白反应抑制了NPC1L1、HMGCR的表达，增加了MTP的表达。相反，在无外源胆固醇的条件下，高浓度的油酸通过折叠蛋白反应增加了NPC1L1、HMGCR的表达。更多还原

【Abstract】 Oleic acid is a main component of dietary oil.It has been shown that Oleic acid is arecognized potent inducer to promote the synthesis and secretion oftriacylglycerol-rich lipoprotein,in which unesterified and esterified cholesterolderived from the plasma membrane were increased in intestinal mucosa epithelialcells incubated with oleic acid.Furthermore,it causes a modest increase of thesynthesis of cholesterol in the absence of micellar cholesterol.It has been reportedthat high concentration of Oleic acid decreases the percentage of cholesterolabsorption in animals fed by high amount of cholesterol.Epidemiological studies hasshown that a certain concentration of oleic acid can reduce the concentration ofplasma LDL cholesterol.So,oleic acid together with polyunsaturated fatty acid isknown as the“healthy fatty acid”.However,the mechanism for how oleic acidinflences the absorption and transport of cholesterol is not clear.Hypercholesterolemia is a significant risk factor for atherosclerosis.In addition tode novo cholesterol synthesis,cholesterol derived from the diet also contributes to theamount of cholesterol circulating in plasma.Moreover,reducing the intestinalabsorption of dietary and biliary cholesterol will decrease plasma cholesterol level.It is clear,therefore,that the intestine plays an important role in maintainingwhole-body cholesterol homeostasis.In the past few years,Niemann-Pick C1-Like1 （NPC1L1）,which resides in the brush-border membrane of intestine cells,has beenidentified as a cholesterol transporter that plays an important role in the absorption ofcholesterol by intestine.The NPC1L1 null mice are completely resistant todiet-induced hypercholesterolemia and do not get hypercholesterolemia in response toa high-cholesterol diet.ABCG5 and ABCG8 also expressed on the brush-border membrane of intestine cells,which function as a heterodimer,regulate the absoptionof cholesterol by facilitating the efflux of cholesterol from absorptive cells back intothe lumen.HMGCR is the rate-limiting enzyme of cholesterol synthesis.Part ofabsorbed and synthesized cholesterol is converted to cholesterol ester by ACAT2 inendoplasmic reticulum.Microsomal triglyceride transfer protein （MTP） is absolutelyessential for the assembly and secretion of triglyceride-rich lipoproteins in theintestine and liver.Sterol regulatory element binding protein-2 （SREBP-2） is amembrane-bound transcription factor that upon proteolytic processing can activate theexpression of genes involved in cholesterol biosynthesis and uptake.It has beenshown to up-regulate the expression of NPC 1 L1 and HMGCR,and negatively regulatethe expression of MTP when SREBP-2 is actived.Endoplasmic reticulum（ER） is the principal site for protein synthesis and folding,biosynthesis of cholesterol and fatty acids,as well as Ca²⁺ storage and signaling.Anyperturbation that interferes with these activities leads to ER stress and activates theunfolded protein response （UPR）,resulting in transcriptional activation of geneswhose products promote the capacity of ER to alleviate the stress.It has beendocumented that OA- induced hepatic ER stress inhibits the secretion ofapolipoprotein B 100.However,whether OA would induce ER stress and further affectthe expression of cholesterol transport-related proteins in the intestinal mucosaepithelial cells is not clear.To explore the effect of OA on the expression of cholesterol transport-relatedprotein in intestinal mucosa epithelial cell and the associated mechanism,CaCo-2cells serve as a model and were incubated with various lipid micelles in this paper.CaCo-2 cells were incubated with various types of micelles containing differentconcentration of oleic acid with or without 0.2mM cholesterol.Western blottinganalysis was applied to determine the protein masses of NPC1L1,ABCG8,BRP78,mature SREBP-2 and ATF6.RT-PCR was used to detect the spliced XBP1、CHOP、NPC1L1、ABCG8、HMGCR、ACAT-2、MTP mRNA.4 - phenyl-butyric acid （PBA） wasemployed to confirm whether the expression variation of cholesterol transport-relatedproteins was induced by UPR or not. The research indicated that both mRNA and protein expression of ACAT-2、SREBP-2 was not altered in CaCo-2 cells incubated with different lipid micelles.Inthe presence or absence of micellar cholesterol,high concentration of oleic acid（0.5mM、1mM ） increased the amount of spliced XBP lmRNA,mature ATF-6 protein,BIP protein and CHOP mRNA expression,resulting in ER stress.In the presence of micellar cholesterol,OA increased the expression of ABCG8protein and MTP mRNA and decreased the expression of NPC1L1 and HMGCR in aconcentration-dependent manner in CaCo-2 cells incubated with OA/CHOL.Incontrast,in the absence of micellar cholesterol,OA increased the expression ofNPC1 L1 and HMGCR in a dose-dependent manner in cells incubated with OA alone.Treatment of CaCo-2 cells with PBA supressed OA-induced incrases of XBP 1 andthe protein mass ofmATF6,as well as BIP and CHOP expression.UPR inhibitor PBA diminished the effect of OA-induced UPR on the expression ofNPC1L1,HMGCR and MTP mentioned above in cells incubated OA/CHOL or OAalone.In a word,in the presence or absence of micellar cholesterol,the expression ofNPC1L1、HMGCR、MTP was diffenently regulaged by oleic acid-inducedendoplasmic reticulum stress in CaCo-2 cells.In the presence of micellar cholesterol,OA-induced UPR increased MTP expression and decreased the expression ofNPC1L1 and HMGCR in a concentration-dependent manner in CaCo-2 cellsincubated with OA/CHOL.In contrast,in the absence of micellar cholesterol,OA-induced UPR increased the expression of NPC1L1 and HMGCR in adose-dependent manner in cells incubated with OA alone.更多还原