中国恒河猴Mamu-B*1703/EGFP在K562和B16细胞中的表达及分布  

Expression and distribution of Chinese rhesus macaque Mamu-B*1703/EGFP in K562 and B16 cells

作　　者：欧阳东云[1,2] 徐丽慧[3] 高琦[1,2] 郭贺[1,2] 何贤辉[1,2] 

机构地区：[1]暨南大学组织移植与免疫实验中心,广州510632 [2]暨南大学再生医学教育部重点实验室 [3]暨南大学生物工程研究所,广州510632

出　　处：《第三军医大学学报》2010年第9期874-878,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(30230350);广东省自然科学基金(8451063201000340);暨南大学人才启动基金(51208004;51208017)~~

摘　　要：目的分析恒河猴主要组织相容性复合体(major histocompatibility complex,MHC)Ⅰ类分子重链的亚细胞分布,建立适于体外研究恒河猴MHCⅠ类分子表达和抗原呈递功能的细胞系。方法将含Mamu-B*1703重链基因真核表达载体pEGFP-N1分别转染K562和B16细胞,细胞收集后以W6/32单克隆抗体或抗Mamu-B*1703抗血清及PE标记二抗染色,利用激光共聚焦显微镜和流式细胞术分析Mamu-B*1703复合体在细胞内和细胞表面的表达。结果在K562和B16细胞的细胞质内均可观察到与Mamu-B*1703重链基因融合表达的绿色荧光蛋白激发后的绿色荧光,即2种细胞均可表达外源Mamu-B*1703重链分子;但通过藻红蛋白标记的抗MHCⅠ类分子复合体抗体染色显示,Mamu-B*1703复合体仅表达于K562细胞表面,而不表达于B16细胞表面。结论Mamu-B*1703重链可与人K562细胞而不能与小鼠B16细胞内的βm轻链结合组装成复合体,表达于细胞表面,提示研究恒河猴的MHCⅠ分子表达应利用人类的细胞株。Objective To analyze the subcellular distribution of Chinese rhesus macaques’ major histocompatibility complex （MHC） class Ⅰ molecules and to prepare a cell line suitable for study on their expression and antigen presentation potential in vitro.Methods An eukaryotic expression vector pEGFP-N1 harboring the Mamu-B＊1703 heavy chain gene was transfected into K562 （human chronic myelogenous leukemia） and B16 cells （mouse melanoma）.The cells were collected and stained with W6/32 monoclonal antibody or Mamu-B＊1703 antiserum followed by PE-conjugated second antibody staining.Distribution of recombinant Mamu-B＊1703 molecules were observed with inverted laser confocal microscopy and further analyzed by flow cytometry.Results Green-fluorescence was observed in the cytoplasm of both K562 and B16 cells after excitation.This indicated that transfected Mamu-B＊1703 heavy chain gene,which was fused with EGFP sequence,could be expressed in both the cells.But only on the surfaces of K562 cells,instead of on that of B16 cells,Mamu-B＊1703 complexes were displayed as indicated by phycoerythrin-conjugated antibodies to MHC class Ⅰ complexes.Conclusion The heavy chain of Mamu-B＊1703 assembles with human β2m molecules in K562 cells to form a complex that is transported onto cell surfaces.Such an assembly or transport process is unlikely to occur in B16 cells.

关 键 词：主要组织相容性复合体Ⅰ类分子 Mamu-B＊1703 中国恒河猴 转染 

分 类 号：R392.13[医药卫生—免疫学] R394.33[医药卫生—基础医学]