【摘要】 目的:研究可溶性小鼠CD83(mCD83)对树突状细胞(DC)表达共刺激分子及共刺激活性的影响。方法:克隆mCD83基因,构建mCD83胞外功能区与人IgG1αFc段融合基因的真核表达载体pmCD83-hIg,并在COS-7细胞中表达可溶性mCD83-hIg融合蛋白。采用ELISA、Western blot和RT-PCR技术检测mCD83-hIg融合基因在COS-7细胞中的表达。用mCD83-hIg处理小鼠DC细胞系(DC2.4)采用流式细胞术检测DC细胞周期、细胞凋亡和共刺激分子CD80、CD86的表达影响;采用混合白细胞培养试验,检测mCD83-hIg对DC2.4刺激同种异基因T细胞增殖及产生IL-2和IFN-γ的影响。结果:酶切和序列测定鉴定显示构建的真核表达载体完全正确;mCD83-hIg对DC2.4细胞周期和细胞凋亡无影响,但可下调DC2.4表面共刺激分子CD80和CD86的表达;mCD83-hIg处理过的DC2.4刺激同种异基因T细胞增殖及产生IL-2和IFN-γ的能力显著下降。结论:mCD83-hIg可抑制DC表达共刺激分子并下调DC共刺激活性,从而抑制同种异基因T细胞增殖和产生细胞因子。更多还原

【Abstract】 Objective:To study the inhibitory effect of soluble mouse CD83(mCD83) on the expression of costimulatory molecules and costimulatory action of dendritic cell to allogenic T cell.Methods:Eukaryotic expression vector pmCD83-hIg, which expressed mouse CD83 extracellular domain and human IgG1α Fc fusion protein(mCD83-hIg), was constructed. Then the plasmid was transfected into COS-7 cell and expressed mCD83-hIg fusion protein in this cell. The expression of CD83 mRNA and mCD83-hIg fusion protein was determined with RT-PCR, ELISA and Western blot. In order to determine the inhibitory effect of mCD83-hIg on the expression of costimulatory molecules on DC, DC2.4 was treated with mCD83-hIg for 72 hour, then the cell cycle and apoptosis of DC2.4 were analyzed by flow cytometry stained with PI, and the expression of costimulatory molecules on DC2.4 was tested by flow cytometery too. The stimulatory action of mCD83-hIg treated DC to the proliferation and secretion of IL-2 and IFN-γ in allogenic T cell were tested with mixed leukocyte culture(MLC) by MTT method and ELISA respectively.Results:The Eukaryotic expression vector, pmCD83-hIg, was constructed successfully determined by endonuclease digested and by sequencing. Treament with mCD83-hIg had no effect on the cell cycle and apoptosis of DC2.4, but it down-regulated the expression of co-stimuatory molecules, CD80 and CD86, which led to the degression of the stimulatory action of DC to allogenic T cells proliferation and secretion of IL-2 and IFN-γ.Conclusion:mCD83-hIg fusion protein could inhibit costimulatory action of DC to allogenic T cell by means of mCD83-hIg inhibited the expression of costimulatory molecules on DC.更多还原