【作者】 肖云玲；

【导师】 高海青；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 第一部分CTLA4Ig诱导T细胞对氧化修饰低密度脂蛋白免疫无能的研究目的:目前研究认为,动脉粥样硬化是一种炎症性疾病,免疫应答是引起机体炎症反应的重要因素。树突状细胞(dendritic cell,DC)、T淋巴细胞和氧化修饰低密度脂蛋白(oxidized-loW density lipoprotein,ox-LDL)均参与了致动脉粥样硬化的免疫反应。本研究拟采用融合蛋白CTLA4Ig阻断DC上B7与T细胞上CD28结合而传递的共刺激信号,诱导T细胞对ox-LDL的免疫无能,并对机理进行初步探讨,以期发现防治动脉粥样硬化的新策略。方法:分离外周血单个核细胞,黏附法分离单核细胞,加入rhGM-CSF(500IU/ml)和rhIL-4(500 IU/ml)培养6天以诱导DC。在DC中分别加入PBS、LDL(10μg/ml)、ox-LDL(10μg/ml)和LPS(30 ng/ml),作用48小时,流式细胞法检测CD86、HLA-DR等细胞标志。ELISA法检测上清中IL-12的含量。取刺激后的DC与同种异体T淋巴细胞行混合淋巴细胞反应(mixed lymphocytereaction,MLR),ox-LDL组分别加入不同浓度的CTLA4Ig(0、0.31、0.62、1.25μg/ml),MTT法检测T细胞的增殖。分别以流式细胞法和酶联免疫斑点(ELISpot)法检测MLR中T细胞CD25的表达、T细胞的凋亡和细胞因子分泌情况。结果:1、LPS和ox-LDL可以促进DC分泌IL-12,较PBS和LDL有显著性差异。CD86(B7)荧光表达率在LPS和ox-LDL10μg/ml刺激后较对照组(PBS组)均有明显升高(P＜0.05)。HLA-DR(MHC-Ⅱ)的表达中也有相同的表现。CD86和HLA-DR的MFI在oxLDL(10μg/ml)刺激后明显高于对照组(P＜0.05)。2、ox-LDL负载的DC可明显刺激MLR中的T细胞增殖,SI与PBS组和LDL组比较,均有显著性差异(P＜0.05)。3、ox-LDL能促进MLR中T细胞分泌IL-2和IFNV(Th1样细胞因子),较PBS组和LDL组明显升高(P＜0.05)。ox-LDL也能促进MLR中T细胞分泌IL-4(Th2样细胞因子),较PBS组和LDL组明显升高(P＜0.05)。4、CTLA4-Ig可抑制ox-LDL激发的同种异体MLR中T细胞增殖反应,与未应用CTLA4Ig组比较有显著性差异(P＜0.05,P＜0.01)。5、CTLA4-Ig可明显抑制ox-LDL激发的同种异体MLR中T细胞CD25的阳性率,与未应用CTLA4Ig组比较有显著性差异(P＜0.05,P＜0.01)。6、应用CTLA4Ig后,淋巴细胞出现一定程度的凋亡,与未用CTLA4Ig比较有显著性差异(P＜0.01)。7、CTLA4Ig减少IL-2和IFN Y的ELISpot斑点数,较未应用组有显著性差异(P＜0.05,P＜0.01);CTLA4Ig增加IL-4的斑点数,较未应用组有显著性差异(P＜0.05)。结论:1、ox-LDL可以促进DC的表型成熟,激发MLR,证明ox-LDL是一种抗原物质:2、ox-LDL负载的DC激发的MLR中既有Th1型免疫应答,也有Th2型免疫应答;3、CTLA4Ig可以在体外诱导T细胞对ox-LDL的免疫无能;4、CTLA4Ig可以促进ox-LDL负载的DC激发的MLR中的Th1/Th2偏移;5、CTLA4Ig可以抑制ox-LDL负载的DC激发的MLR中的T细胞活化;6、CTLA4Ig可以促进ox-LDL负载的DC激发的MLR中的T细胞凋亡。第二部分CTLA4Ig基因转染对树突状细胞针对氧化修饰低密度脂蛋白免疫功能的影响目的:目前研究认为,动脉粥样硬化是一种炎症性疾病,DC与动脉粥样硬化发生和发展的病理过程密切相关,干预DC功能可能成为防治动脉粥样硬化的重要措施。CTLA4Ig可与抗原递呈细胞的B7-1/2分子结合,阻断抗原递呈细胞和抗原特异性T淋巴细胞之间的共刺激信号传递。CTLA4Ig基因转染DC在移植实验中可以诱导机体对移植物抗原的免疫耐受。本研究拟采用人外周血单个核细胞诱导的DC为研究对象,研究含CTLA4Ig基因腺病毒(Ad-CTLA4Ig)转染DC对动脉粥样硬化相关抗原-ox-LDL免疫反应的影响,以期在体外诱导对ox-LDL的免疫耐受,为进一步的动物实验提供基础。方法:分离外周血单个核细胞,黏附法分离单核细胞,加入rhGM-CSF(500IU/ml)和rhIL-4(500 IU/ml)培养4天,用Ad-CTLA4Ig分别以感染指数(multiplicity of infection,MOI)=100,MOI=200和MOI=300感染DC,以含强化绿色荧光蛋白(Ad-EGFP)转染DC和未转染的DC为对照,继续培养4天。ELISA法检测上清CTLA4Ig的含量。荧光显微镜、流式细胞法和免疫印迹(Western blot)法检测CTLA4Ig的表达情况。取转染3天后的DC,加入ox-LDL(10ug/ml),继续培养48hr。ELISA法检测上清IL-12含量。流式细胞法检测CD86和HLA-DR的表达率。取ox-LDL刺激48hr的基因转染DC、未转染DC及加入10ug/mlCTLA4Ig的未转染DC,与同种异体淋巴细胞行MLR,MTT法检测T细胞的增殖。分别以流式细胞法和酶联免疫斑点(ELISpot)法检测MLR中T细胞CD25的表达、T细胞的凋亡和细胞因子分泌情况。结果:1、CTLA4Ig表达率随着时间逐渐增高,在转染后72小时达到高峰。MOI=200时转染率最高。2、CTLA4Ig基因转染DC经ox-LDL刺激后,IL-12的分泌量明显低于Ad-EGFP转染DC(P＜0.05)和未转染DC(P＜0.05)。3、CTLA4Ig基因转染DC CD86和HLA-DR的表达率明显降低,以CD86的降低明显(P＜0.05)。4、CTLA4Ig基因转染DC在MLR中的SI均明显低于未转染DC和Ad-EGFP转染DC(分别P＜0.05)。5、在ox-LDL负载的CTLA4Ig基因转染DC激发的MLR中,CD25的表达率较未转染DC和Ad-EGFP转染DC明显降低(P＜0.05)。6、在ox-LDL负载的CTLA4Ig基因转染DC激发的MLR中,T细胞凋亡率较未转染DC和Ad-EGFP转染DC明显增高(P＜0.01)。7、在CTLA4Ig基因转染DC激发的MLR中,T细胞分泌的IL-2和IFNγ较空白对照和Ad-EGFP转染DC明显降低(均P＜0.05)。T细胞分泌的IL-4较空白对照和Ad-EGFP转染DC明显升高(P＜0.05)。结论:1、Ad-CTLA4Ig转染人DC的最佳MOI=200,最佳表达时相为转染后72小时。2、Ad-CTLA4Ig转染可以降低DC负载ox-LDL后IL-12的分泌,降低CD86和HLA-DR的表达率和刺激MLR的能力,使DC保持一定的未成熟状态。3、Ad-CTLA4Ig转染DC可以在体外诱导对动脉粥样硬化抗原ox-LDL的免疫耐受。4、Ad-CTLA4Ig转染DC通过降低T细胞的激活、促进T细胞凋亡和促进免疫应答的Th1/Th2偏移等机制来诱导对ox-LDL的免疫耐受。更多还原

【Abstract】 PartⅠCTLA4Ig induces immune anergy of T cells to oxidized-low density lipoprotein in vitro.Aim:Recently it is accepted that atherosclerosis is a disease of inflammation,and immune reaction is the most important element in inflammation.The immune elements related to atherosclerosis include dendritic cells(DC),T cells and oxidized-low density lipoprotein(ox-LDL).In this study,CTLA4Ig was used to block the co-stimulation signals between DCs and T cells to induce anergy of T cells to ox-LDL.Methods:Monocytes were separated from peripheral blood by adhension methods and cultured with rhGM-CSF 500IU/ml and rhIL-4 500IU/ml to get DCs.After 6 days culture,cells were treated with PBS,LPS(20ng/ml),LDL(10μg/ml)and ox-LDL(10μg/ml)for 48 hours.Phenotype(CD14,CD86 and HLA-DR)were analysed by flow cytometry(FCM).IL-12 secreted by DCs was assayed by ELISA method.The treated DC were mixed with allogeneic T lymphocytes for further 4 days.CTLA4Ig of different concentration(0、0.31、0.62、1.25μg/ml)were added at the beginning of the mix.The proliferation of Tlymphocytes was analysed by MTT method.Results:1.Ox-LDL stimulated DCs to secret IL-12 more than PBS and LDL significantly.Expression of CD86 and HLA-DR of DCs treated with LPS and ox-LDL were significantly higher than that with PBS and LDL(P＜0.05, respectively).2.DCs treated with LPS and ox-LDL stimulated the allogenic lymphocyte mix reaction significantly than that treated with PBS and LDL(P＜0.05, respectively).3.Ox-LDL and LPS treated DCs stimulated T cells in MLR to secret IL-2,IFNγand IL-4 significantly higher than those of PBS and LDL(P＜0.05, respectively).4.CTLA4Ig suppressed the allogenic lymphocyte mix reaction significantly with significant dose-dependent effect(P＜0.05,P＜0.01,respectively).5. CTLA4Ig inhibited CD25 expression in MLR stimulated by ox-LDL treated DCs significantly compared with that in absence of CTLA4Ig(P＜0.05,P＜0.01, respectively).6.CTLA4Ig induced T cell apoptosis in MLR significantly compared with that in absence of CTLA4Ig(P＜0.01,respectively).7.CTLA4Ig inhibited ELISpot counts of IL-2 and IFNγsignificantly compared with that in absence of CTLA4Ig,while increased ELISpot counts of IL-4(P＜0.05,P＜0.01,respectively).Conclusions:1.Ox-LDL is an antigen.2.Ox-LDL can stimulates both Thl immune reaction and Th2 immune reaction.3.CTLA4Ig can induced T cell anergy to ox-LDL in vitro.4.CTLA4Ig can induce Th1/Th2 deviation in MLR.5.CTLA4Ig can inhibit T cell activation in MLR.6.CTLA4Ig can induce T cell apoptosis in MLR. PartⅡInfluence of CTLA4Ig gene transfection on immune function of DCs to oxidized-low density lipoprotein.Aim:Now it is accepted that atherosclerosis is one kind of inflammation disease, and DCs are concerned with atherosclerosis.CTLA4Ig can combine with B7 on DCs to block costimulation signals conducted between DCs and T cells.In this study,DCs derived from peripheral mononuclear cells were transfected with CTLA4Ig gene by adenovirus,to detect the influence on immune function of DCs to oxidized-low density lipoprotein.Methods:Monocytes were separated from peripheral blood by adhension methods and cultured with rhGM-CSF 500IU/ml and rhlL-4 500IU/ml to get DCs.After 4 days culture,DCs were transfected with adenovirus with CTLA4Ig gene(Ad-CTLA4Ig)at different multiplicity of infection(MOI)(MOI=100,MOI=200 and MOI=300 respectively)and cultured for further 4 days.DCs transinfected with Ad-EGFP and non-transfected DCs were used as controls.CTLA4Ig expression was detected by ELISA,fluorescence microscope,flow cytometry and Western blot respectively. DCs transfected for 3 days were treated with ox-LDL(10ug/ml)for further 48 hours. IL-12 secretion was assayed by ELISA,and phenotypes(CD86,HLA-DR)were tested by flow cytometry.DCs(Ad-CTLA4Ig DC,Ad-EGFP DC,un-transfected DC, un-transfected DC with 10ug/ml CTLA4Ig)treated with ox-LDL were mixed with allogeniec T cells for further 4 days.CD25 expression of T cells in MLR and apoptosis of T cells in MLR were assayed by flow cytometry.Cytokine secretion (IL-2,IL-4 and IFNγ)were assayed by ELISpot.Results:1.The climax of CTLA4Ig expression is at the time after 3 days transfection at MOI=200.2.Secretion of IL-12 of DCs transfected with Ad-CTLA4Ig were lower than those of DCs transfected with Ad-EGFP and un-tranfected significantly(P＜0.05,respectively).3.CTLA4Ig transfection inhibited expression of CD86 and HLA-DR significantly(P＜0.05,respectively).4.SI of MLR stimulated with DC transfected with Ad-CTLA4Ig was inhibited significantly than those of DCs transfected with Ad-EGFP and un-tranfected(P＜0.05,respectively).5.CD25 expression of T cells in MLR stimulated with DCs transfected with Ad-CTLA4Ig was lower than than those of DCs transfected with Ad-EGFP and un-tranfected significantly(P＜0.05,respectively).6.Apoptosis of T cells in MLR stimulated with DCs transfected with Ad-CTLA4Ig was higher than than those of DCs transfected with Ad-EGFP and un-tranfected significantly(P＜0.05,respectively).7.DCs transfected with Ad-CTLA4Ig inhibited seretion of IL-2 and IFNγsignificantly compared with DCs transfected with Ad-EGFP and un-tranfected significantly (P＜0.05,respectively),while stimulated IL-4 secretion significantly(P＜0.05, respectively).Conclusions:1.The appropriate transfection time of Ad-CTLA4Ig on DCs was 3 days,and the appropriate transfection MOI=200.2.Ad-CTLA4Ig transfection decreases expression of CD86 and HLA-DR on DCs,and attenuates function of DCs to stimulate MLR.So Ad-CTLA4Ig transfection retards maturation of DCs.3.DCs tranfected with Ad-CTLA4Ig can induce immune anergy ofT cells to ox-LDU 4.DCs tranfected with Ad-CTLA4Ig induce immune anergy to ox-LDL through mechanisms of inhibiting T cells activation,stimulation of T cells apoptosis and induction of Th1/Th2 deviation.更多还原