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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 type Ⅰ heavy chain in major histocompatibility complex (MHC) of rhesus monkeys and establish a cell line suitable for studying the expression of MHC class Ⅰ molecules and antigen presenting function in rhesus monkey. Methods The eukaryotic expression vector pEGFP-N1 containing the heavy chain gene Mamu-B * 1703 was transfected into K562 and B16 cells. The cells were collected and then harvested with either W6 / 32 monoclonal antibody or anti-Mamu-B * 1703 antiserum and PE labeled secondary antibody Staining, the expression of Mamu-B * 1703 complex in cells and on the cell surface was analyzed by laser confocal microscopy and flow cytometry. Results Green fluorescence after green fluorescent protein fusion with Mamu-B * 1703 heavy chain gene was observed in the cytoplasm of both K562 and B16 cells, that is, both cells could express exogenous Mamu-B * 1703 heavy chain However, staining with phycoerythrin labeled anti-MHC class I antibody showed that the Mamu-B * 1703 complex was only expressed on the surface of K562 cells but not on the surface of B16 cells. Conclusion The heavy chain of Mamu-B * 1703 can be assembled with human K562 cells but not with the βm light chain of mouse B16 cells to form a complex that is expressed on the cell surface, suggesting that expression of MHC I molecules in rhesus monkeys should be performed using human cells Strain.