摘要
目的:应用人重组真核表达载体pMAGE-A1/EGFP转染的树突状细胞(DCs)在体外诱导MAGE-A1特异性T 细胞免疫。方法:构建重组质粒pMAGE-A1/EGFP,体外电转染该质粒入DCs,通过流式细胞仪检测和胞内染色法LDH法比较转染前后DCs在蛋白表达、细胞成熟度和诱导MAGE-A1特异性T细胞免疫能力的差异。结果:成功构建pMAGE-A1/EGFP 真核表达质粒,电穿孔法导入DCs后观察到MAGE-A1与EPFG实现等效表达,分别为8．8％±0．9％和8．47％±0．78％。 pMAGE-A1/EGFP转染的DCs表面CD86、CD40L和CD83表达水平显著升高,提示DCs的成熟度增加;同时表达的MAGE-A1 蛋白可与小鼠来源抗MAGE-A1的单抗发生特异性结合,并在体外诱导MAGE-A1特异性CD4+T细胞克隆扩增,当再次接触 MAGE-A1抗原时大量分泌IFN-γ。结论:pMAGE-A1/EGFP转染的DCs不仅能在体外诱导MAGE-A1特异性T细胞免疫,还可通过流式细胞仪进行快速检测,为临床开展MAGE-A1为基础的免疫治疗奠定了一定基础。
Objective：To induce MAGE-A1 specific T cell immunity by dendritic cells（DCs） transfected with recombinant human eukaryotic vector pMAGE-A1/EGFP. Methods ：The recombinant human eukaryotic vector pMAGE-A1/EGFP was constructed and transfected into human PBMC-derived DCs. Using FACS analysis and intracellular staining methods,differences in proteins expression, maturation and capacity to induce MAGE-A1 specific T cell immunity of DCs before and after transfection were identified. Results： Successfully the recombinant human eukaryotic vector pMAGE-A1/EGFP was constructed and equally MAGE-A1 and EGFP was expressed after transfection into DCs by electroporation,which was 8.8%±0. 9% and 8. 47%±0. 78% respectively. After transfection, the expression of CD86,CIMOL and CD83 were upregulated,which suggested that maturation of DCs was enhanced. MAGE-A1 expressed on the surface of transfected DCs could be combined with anti-MAGE-A1 monoclonal antibody specially, and indued amplification of MAGE-A1 specific CD4^＋ T cell clones,which secreted more IFN-γ when contacting with MAGE-A1 antigen once again. Conclusion：As a kind of promising vaccine, DCs transfected with human eukaryotic vector pMAGE-A1/EGFP could not only induce MAGE-A1 specific T cell immunity in vitro,but also be detected by FACS rapidly as well,thus will give hope to immunotherapy based on MAGE-A1.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2006年第1期49-52,57,共5页
Chinese Journal of Immunology
关键词
重组真核表达载体
树突状细胞
MAGE-A1
EGFP
特异性T细胞免疫
Recombinant human eukaryotic vector
Dedritic cells
Melanoma antlgen-encodlng gene-A1 （ MAGE-A1 ）
Enhanced green fluorescent protein（EGFP）
Specific T cell immunity
