从K562细胞中提取热休克蛋白GP96及其对人树突状细胞分化和功能的影响

Differentiation and Function of Human Dendritic Cells Influenced by Heat Shock Protein GP96 Purified from K562 Cells

本研究旨在建立从K5 6 2细胞系中提取热休克蛋白GP96的方法 ,进而研究GP96对人树突状细胞分化和功能的影响。采用蛋白分离提纯技术 ,将K5 6 2细胞裂解 ,经饱和硫酸铵沉淀、亲和层析、离子交换层析后提纯出热休克蛋白GP96 ,用SDS PAGE凝胶电泳和Westernblot方法对其进行鉴定。将提纯的GP96加入从正常人外周血单个核细胞诱导培养的树突状细胞 (dendriticcell,DC)中 ,通过流式细胞术检测DC细胞表型变化 ,用MTT法测定混合淋巴细胞反应 (mixedlymphocytereaction ,MLR)。结果表明 :从 1× 10 1 0 K5 6 2细胞中能提纯出热休克蛋白GP96 6 0 - 80 μg ;加入GP96制备的HSP DC ,其细胞表面标志CD83、CD86和HLA DR的表达率比常规培养DC明显升高 ,CD1a表达率降低 (P <0 .0 5 ) ;HSP DC比常规培养DC具有更强的刺激混合淋巴细胞反应的能力 (P <0 .0 5 )。结论 :从K5 6 2细胞中可以提取出热休克蛋白GP96 ;热休克蛋白GP96可以促进树突状细胞分化成熟 。

This study was to establish the method of purifying heat shock protein GP96 from K562 cells and explore the differentiation and function of human DC influence d by heat shock prolein (HSP). Using ammonium sulfate precipitation, conA-seph arose affinity chromatography and DEAE-sephacel anion exchange chromatography GP96 fr om K562 cells lysate was isolated and purified. The identification of the purifi ed protein was controlled by Western blot with anti-human GP96 IgG. Human dendritic cell derived from per iphera l blood mononuclear cell were cultured with purified GP96. The phenotype changes of DC were analyzed by flow cytometry and MLR was detected by MTT. The result s showed that 60-80 μg GP96 was purified successfuly from 1×10 10 K562 ce lls. DC stimulated with HSP-GP96 had higher expression rates of CD83, CD86, HLA-DR and lower expression rates of CD1a and had stronger ability to ind u ce T cells proliferation. It is concluded that heat shock protein GP96 can be i solated and purified from K562 cells and could induce maturation of dendritic c el l. HSP-DC vaccine show stronger ability to induce T cell proliferation than DC.