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目的构建人CD83全长编码基因转染293细胞,并探讨其对单核细胞的作用。方法 RT-PCR方法从成熟人树突状细胞(DC)扩增出CD83 cDNA,插入pIRES2-EGFP载体,脂质体转染293细胞,筛选出稳定表达目的基因细胞株;从人外周血单个核细胞(PBMC)中磁珠分选单核细胞,LPS刺激的同时加入293/CD83细胞或293/mock,24 h后检测细胞活化状态以及培养上清中TNF-α水平。结果经流式细胞术反复检测绿色荧光蛋白(GFP)报告基因及CD83表达水平,筛选获得获得一株稳定表达膜型CD83分子的基因转染细胞株293/CD83;体外实验显示293/CD83可促进LPS刺激的单核细胞活化水平和TNF-α分泌量。结论成功构建表达人CD83分子的293细胞株,293/CD83对LPS刺激的单核细胞有协同刺激作用。
Objective To construct human CD83 full-length gene and transfect it into 293 cells and investigate its effect on monocytes. Methods CD83 cDNA was amplified by RT-PCR from mature human dendritic cells (DCs) and inserted into pIRES2-EGFP vector. The liposomes were transfected into 293 cells and the stable cell lines were selected. Monocytes were sorted by magnetic beads in PBMCs. 293 / CD83 cells or 293 / mock cells were simultaneously stimulated with LPS. After 24 h, the cell activation status and the level of TNF-α in the culture supernatants were detected. Results Green fluorescent protein (GFP) reporter gene and CD83 expression level were detected by flow cytometry repeatedly. A gene-transfected cell line 293 / CD83 stably expressing membrane-type CD83 molecule was screened. In vitro experiments showed that 293 / CD83 could promote LPS-stimulated monocyte activation and TNF-α secretion. Conclusion The 293 cell line expressing human CD83 molecule was constructed successfully, and 293 / CD83 synergistically stimulated LPS-stimulated monocytes.