多发性硬化抗原特异性CD4^＋CD25^＋调节性T细胞的体外扩增及其功能效应.pdf

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1、·基础论著·多发性硬化抗原特异性CD4+CD25+调节性T细胞的体外扩增及其功能效应姜红刘广志杨亭亭何洋于鑫向雅娟高旭光【摘要】目的针对多发性硬化(MS)患者CD4CD25调节性T细胞(Treg)的免疫功能缺陷，尝试利用体外培养法获取功能正常的Treg。方法密度梯度离心法获得MS患者(=5)和健康对照者(=5)外周血单个核细胞(PBMC)，免疫磁珠法分选B细胞和CD4CD25一初始T细胞，前者进一步经可溶性CD40配体(sCD40L)刺激活化后作为抗原提呈细胞(APC)，与后者混合体外共培养，并以髓鞘碱性蛋白(M

2、BP)85．。。等刺激后收获细胞；经流式细胞仪分选获得CD4CD25highCD127Treg。通过增殖分析鉴定所获Treg的免疫抑制功能。结果磁珠法分选B细胞、初始T细胞的纯化率分别达到90％和80．62％。体外诱导扩增所获的MBP反应性Treg比例为3．5％～6％，MS组与健康对照组Treg比较抑制功能活性无明显差异。结论经此方法可扩增获得免疫功能正常的Treg细胞，进而为今后开展MS的特异性免疫调节治疗提供客观的依据。【关键词】多发性硬化；T淋巴细胞，调节性；体外培养Expansionandfunction

3、analysisofantigen-specificCD4CD25htghregulatoryTcellinpatientswithmul~plesclerosisJiangHong,LiuGuangzhi,YangTingting,HeYang,YuXin,XiangYajuan，GaoXuguang．DepartmentofNeurology,PeMngUniversityPeople'sHospital,Beijing100044,ChinaCorrespondingauthor：GaoXuguang,Em

4、ail：jh1991@sina．com[Abstract]ObjeetiveToobtamnormalregulatoryTcells(Treg)throughinvitrocultureinpatientswithmultiplesclerosis(MS)demonstratingimmunedefectofCD4CD25Treg．MethodsPeripheralbloodmononuclearcells(PBMC)ofMSpatientsandhealthycontrolwasisolatedbydensi

5、tygradientcentrifugation，andCD4CD25一naiveTceUsandaUogeneicBcellswere~thersortedwithimmunomagneticbeads．AllogeneicBcell，afteractivationwithsCD40L，wasco—culturedwithCD4CD25一naiveTcellsinthepresenceofmyelinbasicprotein(MBP)85．99peptide，Rapamycinand4-1BBLfor10day

6、s．CD4CD25~hcD127Tregweresortedwithflowcytometry，anditsimmunosuppressivefunctionwasidentifiedbyproliferationassay．ResultsPurificationrateofCD4CD25一naiveTceilsandallogeneicBcellsthroughirrmmomagneticbeadswere90％and80．62％aswellasforMBP—reactiveTreg3．5％-6％through

7、／nvitroculture．TheTregimmunoactivitiesdemonstratednosignificantdiferencecompared、vitbthatofhealthycontro1．ConclusionTregwithnormalimmunoactivitiescallbeobtainedthroughthiscocultu~methodinvitro，andwilprovidereliableevidenceforflLrtht~"antigen-specificimmunomod

8、ulatorytherapy．[Keywords]Multiplesclerosis；T-lymphocytes，regulatory；／nvitroculture多发性硬化(multiplesclerosis，MS)是一种调节性T细胞(Treg)的效应功能较正常对照下降[1】。发生于中枢神经系统(CNS)的炎性脱髓鞘疾病，多项动物实验发现，经静脉注射给予实验性自身免多见