异种蛋白联合重组人粒／巨噬细胞集落刺激因子和卡介苗免疫调节效应的体外研究

本研究探讨异种蛋白neu-Fc协同重组人粒/巨噬细胞集落刺激因子(GM-CSF)及卡介苗(BCG)对Th1型和Th2型免疫应答的调节作用。通过基因工程技术将大鼠neu L2-S2功能域与人IgG Fc区编码序列融合,构建真核表达载体。在CHO细胞中获得稳定表达,应用rProtein A Sepharose Fast Flow亲和层析纯化neu-Fc融合蛋白,应用SDS-PAGE和Western blot鉴定重组蛋白。Ficoll法分离人外周血单个核细胞(PBMNC),MTT法检测融合蛋白对PBMNC的促增殖作用。通过ELISA法检测IL-12和IL-10水平的变化。结果表明:PBMNC经MCF-7细胞上清诱导后,IL-12水平降低(p<0.05),IL-10水平显著升高(p<0.01)。10nmol/L neu-Fc对PBMNC具有显著的促增殖作用。与GM-CSF、BCG或者neu-Fc单独作用相比,neu-Fc与GM-CSF、BCG联合作用可显著上调IL-12表达,下调IL-10表达(p<0.01)。结论:neu-Fc具有很好的促增殖作用,GM-CSF和BCG协同neu-Fc能有效诱导Th1型细胞应答。

Immunoregulation Effects In Vitro of the Xenoprotein in Combination with Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor and Bacillus Calmette-Guerin

This study was aimed to investigate the effects of xenogeneic antigen neu-Fc in combination with the recombinant human granulocyte-macrophage colony stimulating factor(GM-CSF) and Bacillus Calmette-Guerin(BCG) on the regulation of Th1 and Th2 immune response in vitro.The rat neu L2-S2 domain was engineered as a chimeric protein with human IgG Fc.The eukaryotic expression vector was constructed.The recombinant protein was stably expressed in CHO cells and purified by rProtein A Sepharose Fast Flow column.The recombinant protein was identified by SDS-PAGE and Western blot.Peripheral blood mononuclear cells(PBMNCs) were obtained by means of standard Ficoll separation from the blood of healthy donors.Neu-Fc-induced PBMNC proliferation was tested by MTT.The production of IL-12 and IL-10 was measured by ELISA.The results showed that the level of IL-12 decreased and IL-10 increased after PBMNCs were incubated with MCF-7 cultural supernatant.10 nmol/L neu-Fc strongly induced the cell proliferation.Compared with neu-Fc or GM-CSF or BCG treatment alone,neu-Fc in combination with GM-CSF and BCG significantly stimulated IL-12 production and inhibited IL-10 production(p<0.01).It is concluded that the neu-Fc can stimulate the proliferation activity of PBMNCs.neu-Fc,GM-CSF and BCG costimulation efficiently induces Th1 immune response.