摘要：
背景 以往对组织中抗原递呈细胞(APCs)的研究多采用体外免疫组织化学的方法,其结果易受到多种因素的影响,曾有研究者采用前房注射荧光素标记抗体的方法对眼前节组织中的APCs进行研究,但易损伤角膜等组织.采用活体标记的方法探讨虹膜和睫状体中APCs的表型特征,对进一步阐明APCs在维持眼部免疫平衡状态中的作用及机制具有重要意义. 目的 探讨正常小鼠虹膜中APCs的表型特征、分布和形态学特点.方法 6～8周龄的SPF级雌性BALB/c小鼠51只按照随机数字表法分为17个组,在生物解剖显微镜下用玻璃微量注射针于角巩膜缘后0.5 mm处刺入玻璃体腔内注射2.0 μl AlexaFluor594或Alexa Fluor 488标记的卵清蛋白(OVA)、抗CD11c、主要组织相容性复合体分子Ⅱ(MHC-Ⅱ)、F4/80、B7-1和B7-2单克隆抗体或其两两抗体的混合液,注射24 h后取小鼠虹膜组织,结合平片技术和激光扫描共焦显微镜观察虹膜中APCs的表型特征、分布及形态学特点,并用体外染色实验验证体内染色结果. 结果 正常小鼠虹膜中存在大量呈规则网络状分布的OVA+、F4/80+、CD11c+、MHC-Ⅱ+、B7-1+和B7-2+细胞,Alexa Fluor 594标记的阳性细胞呈红色荧光,Alexa Fluor 488标记的阳性细胞呈绿色荧光.双重染色实验结果可见,虹膜中约有90％的F4/80+细胞为OVA+,约有60％的F4/80+细胞和CD11c+细胞表达MHC-Ⅱ,约有35％的F4/80+细胞和CD11c+细胞同时表达B7-1和B7-2,70％以上的OVA+细胞为MHC-Ⅱ+.根据标记细胞的形态不同可分为树突状细胞(DC)和多形性细胞两大类.结论 活体玻璃体腔内注射荧光素标记抗体的方法可以准确观察正常小鼠虹膜中的APCs.虹膜中APCs的表型特征、分布和形态学特点与眼免疫偏离和炎症密切相关.%Background The conventional study of antigen-presenting cells(APCs)in eye relies on in vitro histoimmunochemistry,but its outcome is influenced by many factors.The anterior chamber injection of fluoresceinmarked antibody was used as a new approach before,however,it is liable to lead to injury of cornea.The intravitreal injection of fluorescein-labeled antibody may be important for the in vivo study of the phenotype features of APCs in iris,which is significant for evaluating the function of APCs in immune homeostasis.Objective This study was to investigate the phenotype characters,distribution and morphology of different types of APCs in the normal murine iris.Methods Fifty-one SPF female BALB/c mice(from 6-to 8-week old)were randomized into 17 groups according to the injection of different antibodies.Alexa Fluor 594 or Alexa Fluor 488-tagged ovalbumin (OVA),CD11 c,major histocompatibility complex Ⅱ (MHC-Ⅱ),F4/80,B7-1 and B7-2 monoclonal antibodies or mixtures of two antibodies (2.0 μl)were intravitreally injected at 0.5 mm far from corneal limbus with microneedle under the biomicroscope.The iris tissues were isolated 24 hours after injection.The phenotype characters,precise distribution and morphology of different types of APCs were identified by epifluorescence microscope and laser confocal microscope.In vitro staining was also performed to validate the in vivo staining results.Results After in vivo staining via intravitreal injection,the cell positive for OVA as well as MHC-Ⅱ,F4/80,CD11 c,B7-1 and B7-2 were exhibited with the regular networkline appearance throughout the normal murine iris.Positive cells tagged with Alexa Fluor 594 or Alexa Fluor 488 presented the red or green fluorescence.Double-fluorescein staining showed that about 90％ of F4/80+ cells were OVA+,and MHC-Ⅱ was expressed in about 60％ of F4/80+ cells and CD11c+cells,and about 35％ of F4/80+ cells and CD1 1 c+ cells expressed B7-1 and B7-2 simultaneously,and over 70％ of OVA+ cells were positive to MHC-Ⅱ.These labeled cells were identified as two populations based on their shape.One type was dendritiform cell (DC) with a small cell body and many long dendrites,including OVA+,CD1 1 c+,F4/80+ cells and MHC-Ⅱ + cells ; and the other types were polymorphic population being round,pleomorphic or irregular shape with a large cell body and a few short dendrities,including B7-1 + and B7-2+ cells.Conclusions In vivo intravitreal injection of labeled antibodies can be adapted to visualize the labeled cells in the murine iris.APCs with distinct morphologies,phenotypes and distribution may contribute to the immunologically privileged feature and inflammation of the eye.
