MHC-Ig/肽复合体在实验性自身免疫性葡萄膜炎CTLs研究中的应用  

Application of MHC-Ig/Peptide Polymer in Detecting Antigen-specific Cytotoxic T Lymphocytes in Mice with Experimental Autoimmune Uveitis

作　　者：雷松[1,2] 岳利民[1] 毛咏秋[3] 杨莉[3] 

机构地区：[1]四川大学华西基础医学与法医学院生理学教研室,成都610041 [2]四川大学华西医院病理科,成都610041 [3]四川大学华西医院生物治疗国家重点实验室,成都610041

出　　处：《四川大学学报（医学版）》2010年第1期35-38,共4页Journal of Sichuan University(Medical Sciences)

摘　　要：目的探讨MHC-Ig/肽复合体用于小鼠实验性自身免疫性葡萄膜炎(EAU)特异性细胞毒性T淋巴细胞(CTLs)研究的应用价值。方法用人类光感受器间维生素A类结合蛋白(IRBP)合成多肽IRBP161-180免疫B10RⅢ小鼠制备EAU动物模型。将源于IRBP161-180多肽序列的10种不同短肽片段与MHC-Ig多聚体形成复合体,用于检测B10RⅢ小鼠EAU中特异性CTLs。比较各种MHC-Ig/IRBP短肽复合体与CTLs的结合能力,以及刺激CTLs的增殖和IFN-γ的表达水平,并选用对CTLs作用最强的复合体,检测小鼠在EAU不同发病期外周血CTLs的表达情况。结果各MHC-Ig/IRBP短肽复合体能够检测出抗原特异CTLs,其中MHC-Ig/IRBP168-177短肽复合体检测出CTLs的阳性率达12.3%;并且其刺激CTLs的增殖和IFN-γ的表达水平明显高于其它短肽组(P<0.01),可初步推断短肽序列IRBP168-177为该EAU模型特异CTLs主要的抗原识别表位。用MHC-Ig/IRBP168-177短肽复合体检测EAU疾病过程中外周血CTLs的表达发现,EAU在炎症急性期特异性CTLs的阳性表达最高(4.9%±1.1%)。结论MHC-Ig/肽复合体技术,是一种新的定量分析抗原特异性CTLs的方法,能检测抗原特异性CTLs的表达情况,分析CTLs的抗原识别表位,为动物模型EAU的研究提供了高效、特异、敏感的检测手段。Objective To explore the application of MHC-Ig/peptide polymer technique for detecting antigen- specific cytotoxic T lymphocytes（CTLs）in mice with experimental autoimmune uveitis （EAU）. Methods B10RⅢ mice were immunized with an interphotoreceptor retinal-binding protein （IRBP） synthetic peptide （IRBP161-180）. The in vivo primed T cells were separated and stained with MHC-Ig polymer combined with a panel of truncated pcptides derived from IRBP161-180. The level of IRBP-specific CTLs cells was determined by FACS analysis. The CDS＋T cells were isolated from the primed T cells and stimulated with complex polymers containing MHC-Ig and various IRBP-derived peptides. The proliferation of CD8＋ T cells was measured by 3 H thymidine incorporation. The production of interferon- （IFN-） in the cell suspensions was measured by ELISA. Results The IRBP-specific CTLs were detected by MHC-Ig/peptide polymers. The MHC-Ig/IRBP168-177 peptide polymer dtected 12.3% specific CTLs, showing greater ability in stimulating proliferation of CTLs and production of IFN-than the other MHC-Ig/ peptide polymers （P〈0.01）. The truncated 10-mer peptide, IRBP168-177, was the major antigenic epitope for the IRBP-specific CTLs. The MHC-Ig/IRBP168-177 peptide polymer detected the highest level（4. 9± 1. 1%）of specific CTLs from peripheral blood mononuclear cells（PBMCs） at the acute stage of EAU. Conclusion The MHC-Ig polymer technique is an effective instrument for detecting antigen-specific CTLs, with good sensitivity and specificity in EAU studies.

关 键 词：MHC-Ig/肽复合体 实验性自身免疫性葡萄膜炎 细胞毒T细胞 

分 类 号：R773.9[医药卫生—眼科]