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1、华中科技大学硕士学位论文葡萄糖对巨噬细胞泡沫化影响之初步研究姓名:严同申请学位级别:硕士专业:内科学(内分泌)指导教师:高峰20090501华中科技大学硕士学位论文华中科技大学硕士学位论文3葡萄糖对巨噬细胞泡沫化影响之初步研究葡萄糖对巨噬细胞泡沫化影响之初步研究硕士研究生:严同导师:高峰副教授摘摘要要【背景】大血管并发症是导致糖尿病患者死亡和伤残的主要原因。大血管并发症主要表现为大血管动脉粥样硬化。动脉粥样硬化典型的病理表现是巨噬细胞源性泡沫细胞的形成。而导致巨噬细胞泡沫化的主要原因就是过多胆固醇在细胞内的堆积。生理情况下,巨噬细胞通过清道夫受体CD36摄取富含胆固醇的oxLDL,另外一方面,
2、又通过ABCA1将细胞内的胆固醇外流至apoA-I,从而避免了过多胆固醇在细胞内的堆积。巨噬细胞对oxLDL摄取增加或细胞内胆固醇流出减少都能导致泡沫细胞的形成。虽然目前研究发现高血糖是导致动脉粥样硬化的一个独立危险因素,目前对于高血糖和巨噬细胞泡沫化之间的具体关系还不清楚。【方法】分离小鼠原代腹腔巨噬细胞,在5.6mmolL葡萄糖浓度的DMEM培养基中培养24小时,待细胞贴壁后换液,分别将细胞置于含葡萄糖5.6mmolL、16.7mmolL和30mmolL的DMEM培养基中培养。其中5.6mmolL为正常糖浓度对照组,16.7mmolL和30mmolL组为高糖组。培养24小时、48小时和72
3、小时后,收集细胞,提取总RNA,逆转录,使用半定量RT-PCR的方法测定ABCA1和CD36的mRNA表达。【结果】16.7mmolL和30mmolL高糖组24小时、48小时和72小时三个时间点CD36mRNA表达与正常糖浓度对照组相比没有变化(P=0.645)。16.7mmolL高糖组和正常糖浓度对照组相比,24小时、48小时和72小时ABCA1mRNA表达没有差异(P=0.298);30mmolL高糖组和正常糖浓度对照组相比,30mmolL葡萄糖组的ABCA1mRNA表达在24小时出现下降的趋势(P=0.14);在48小时30mmolL葡萄糖组ABCA1mRNA表达较同时间点正常糖浓度对照
4、组下降了40.4%(P=0.009),在华中科技大学硕士学位论文华中科技大学硕士学位论文472小时30mmolL葡萄糖组ABCA1mRNA表达较正常糖浓度对照组下降了48.1%(P=0.015)。【结论】高浓度葡萄糖不影响巨噬细胞清道夫受体CD36表达,但是高糖环境能抑制巨噬细胞ABCA1的表达;因此,在不影响巨噬细胞对oxLDL摄取的情况下,推测葡萄糖可能通过影响胆固醇从巨噬细胞的流出而加速巨噬细胞的泡沫化,从而诱导动脉粥样硬化病变的发生。【关键字】糖尿病;动脉粥样硬化;清道夫受体;CD36;ABCA1华中科技大学硕士学位论文华中科技大学硕士学位论文5EffectsofGlucoseonMa
5、crophageFoamCellationAPreliminaryStudyPostgraduateStudent:YanTongSupervisor:Prof.GaoFengAbstractBackgroundMacrovascularcomplicationsarethemaincauseofmortalityandmorbidityindiabeticpatients.Diabeticmacrovascularcomplicationsmanifestasatherosclerosis.Thehallmarkofatherosclerosisismacrophage-derivedfoa
6、mcellationinsub-endothelialspaceofatherosclerosis-pronearteries.Excessivecholesterolstoredinmacrophagescontributestomacrophagefoamcellation.PhysiologicallymacrophagestakeupoxLDLviascavengerreceptorCD36expressingoncellmembrane.MeanwhilecholesterolstoredinmacrophagesistransportedtoapoA-IviaABCA1.There
7、forethebalancebetweenCD36andABCA1keepsmacrophagesawayfromcholesteroloverload.BothenhancedoxLDLuptakeanddecreasedcholesteroleffluxcaninducemacrophagefoamcellation.Therelationshipbetweenhyperglycemiaandmacrophagefoamcellationremainstobeelucidatedalthoughmanystudieshavedemonstratedthathyperglycemiaisan
8、independentriskfactorforatherosclerosis.sFreshlyharvestedmouseprimaryperitonealmacrophageswereincubatedinDMEMcontaining5.6mmolLglucosefor24hoursat37.AfterreplacingthemediumwhencellswereadherenttocellcultureflaskscellswerefurtherculturedinDMEMcontainingdifferentglucoseconcentrations5.6mmolL16.7mmolLa
9、nd30mmolLglucoserespectively.Tobespecific5.6mmolLservedascontrolgroupand16.7mmolLand30mmolLservedashighglucosegroup.CellswerelysatedfortotalRNApreparationafter24hours48hoursand72hoursrespectively.ABCA1mRNAandCD36mRNAexpressionwereuatedbysemi-quantitativeRT-PCR.ResultsCD36mRNAexpressionat24hours48hou
10、rsand72hoursdidntchangesignificantlyamong16.7mmolLglucosegroup30mmolLglucosegroupandcontrol华中科技大学硕士学位论文华中科技大学硕士学位论文6group(P=0.645).ThemRNAexpressionofABCA1didntshowanyobviouschangesbetween16.7mmolLglucoseandcontrolgroupat24hours48hoursand72hours(P=0.298).ComparedwithcontrolgrouptheABCA1mRNAexpressio
11、nof30mmolLglucosegroupshowedatendencyofdownregulationat24hours(P=0.14)butasignificantdecreaseby40.4%at48hours(P=0.009)furthersuppressionby48.1%at72hours(P=0.015).ConclusionsThepresentstudyshowedthatahighglucoseenvironmentapparentlyhadnoeffectonmacrophagescavengerreceptorCD36mRNAexpressionbutsuppress
12、edmacrophageABCA1mRNAexpression.HencehyperglycemiamayacceleratethepathogenesisofatherosclerosisbyinhibitingmacrophagecholesteroleffluxviaABCA1withoutenhancingCD36mediatedmacrophageoxLDLuptake.KeyWordsdiabetesatherosclerosisscavengerreceptorCD36ABCA1华中科技大学硕士学位论文华中科技大学硕士学位论文1英文缩写词英文缩写词DECODEDiabetesEp
13、idemiology:CollaborativeAnalysisOfDiagnosticCriteriainEurope糖尿病流行病学:欧洲糖尿病诊断标准协作分析研究DCCTDiabetesControlandComplicationsTrial糖尿病控制和并发症研究LDLlowdensitylipoprotein低密度脂蛋白DCCTEDICDiabetesControlandComplicationsTrialEpidemiologyofDiabetesInterven-tionsandComplications糖尿病控制和并发症研究糖尿病干预和并发症流行病学研究UKPDSUnitedKin
14、gdomProspectiveDiabetesStudy英国糖尿病前瞻性研究POPADADThePreventionofProgressionofArterialDiseaseandDiabetesTrial无症状性糖尿病动脉疾病进展预防研究JPADJapanesePrimaryPreventionofAtheros-clerosiswithAspirinforDiabetes日本阿司匹林的糖尿病动脉粥样硬化一级预防研究HDLhighdensitylipoprotein高密度脂蛋白oxLDLoxidizedlowdenselipoprotein氧化低密度脂蛋白SRscavengerrecept
15、or清道夫受体VCAM-1vascular-celladhesionmolecule-1血管细胞黏附分子-1AcLDLacetylatedlowdensitylipoprotein乙酰化低密度脂蛋白AGEsadvancedglycationendproducts高级糖化终产物ROSreactiveoxygenspecies活性氧簇ACATacylCoA-cholesterolacyltransferase酰基辅酶A-胆固醇酰基转移酶ABCA1ATP-bindingcassettetransporterA1ATP结合盒转运体A1ABCG1ATP-bindingcassettetransporte
16、rA1ATP结合盒转运体G1apoA-IapolipoproteinA-I载脂蛋白A-ILCATlecithin-cholesterolacyltransferase卵磷脂-胆固醇酰基转移酶RCTreversecholesteroltransport胆固醇逆转运SR-BIscavengerreceptorclassBtypeI清道夫受体BIAngIIangiotensinII血管紧张素II华中科技大学硕士学位论文华中科技大学硕士学位论文2Fra2fos-relatedantigen2fos相关抗原2CRPCreactiveProteinC反应蛋白ERKextracellularsignal-regulatedkinases细胞外信号调节激酶IL-1interleukin-1白细胞介素1NF-Bnuclearfactor-light-chain-enhancerofactivatedBcells活化B细胞核因子轻链增强子TNF-tumornecrosisfactor肿瘤坏死因子PKCproteinkinaseC蛋白激酶C独创性声明独创性声明本人郑重声明,本学位论文