HEK293T细胞表达的单核细胞趋化蛋白1促进巨噬细胞迁移  

The recombinant monocyte chemotactic protein 1 expressed in HEK293T cells promotes migration of macrophages

作　　者：王媛媛[1,2] 赵俊龙[2] 刘娟[2] 姜亚丽[1,2] 韩骅[2] 秦鸿雁[2] 刘晓渭[1] 

机构地区：[1]第四军医大学附属西京医院肾脏内科,陕西西安710032 [2]第四军医大学基础部医学遗传与发育生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2016年第2期149-152,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81370811)

摘　　要：目的构建巨细胞病毒(CMV)启动子驱动的单核细胞趋化蛋白1(MCP-1)真核表达载体,转染人胚肾HEK293T细胞,验证其对巨噬细胞的趋化作用。方法小鼠基因组DNA作模板,用PCR法获得MCP-1启动子区基因片段,插入p FLAGCMV1载体。用双酶切法和测序鉴定正确后,将构建好的病毒载体转染HEK293T细胞,用Western blot法检测MCP-1的表达,用Transwell^(TM)实验检测转染后细胞分泌的MCP-1对巨噬细胞的趋化作用。结果重组载体酶切鉴定在相应位置有目的片段,转染单核细胞趋化因子重组载体后的HEK293T细胞有MCP-1表达,转染后HEK293T细胞分泌的MCP-1明显增加巨噬细胞的迁移。结论HEK293T细胞表达的MCP-1明显增强巨噬细胞的迁移。Objective To construct the eukaryotic expression vector of monocyte chemokine protein 1（ MCP-1） driven by the cytomegalovirus（ CMV） promoter,transfect the vector into HEK293 T cel s,and detect its chemotaxis to macrophages.Methods MCP-1 promoter was obtained from mouse genome by PCR,and inserted into the vector named p FLAG-CMV1.We validated it by double-enzymes digestion and sequencing. Then HEK293 T cells were transfected with p FLAG-CMV1-MCP-1. The expression of MCP-1 was detected by Western blotting. Finally,we identified the chemotaxis of the recombinant vector to macrophage by Transwell^TMassay. Results The recombinant vector could generate target fragment by double enzyme digestion. HEK293 T cells expressed MCP-1 after they were transfected with the recombinant vector,which increased the migration of macrophages. Conclusion MCP-1 expressed by HEK293 T cells could apparently increase the migration of macrophages.

关 键 词：巨噬细胞 单核细胞趋化蛋白1(MCP-1) 真核表达载体 载体构建 

分 类 号：R392-33[医药卫生—免疫学] R392.12[医药卫生—基础医学]