GM-CSF重组腺病毒转染外周血树突状细胞的研究

目的:利用GMCSF重组腺病毒转染外周血单核细胞,诱导培养树突状细胞(dendriticcell,DC),并与细胞因子培养的DC进行生物学特性的比较。方法:将GMCSF重组腺病毒转染正常人外周血单核细胞,或用重组细胞因子GMCSF,诱导培养获得DC,通过相差显微镜观察DC表面形态变化,FACS分析GMCSF基因转染对DC表面免疫分子表达的影响,Westernblot鉴定GMCSF的表达,ELISA检测IL12分泌水平,MTT法检测DC激发同种异体T细胞增殖的能力。结果:GMCSF重组腺病毒转染外周血单核细胞,能扩增获得DC,该DC高表达CD1a、HLADR、CD80和CD86等免疫分子,并表达细胞因子GMCSF和IL12,对同种异体淋巴细胞有更强的刺激活性。结论:用GMCSF重组腺病毒可从外周血单核细胞中扩增到DC,为DC瘤苗临床应用提供实验基础。

Study of dendritic cell derived from monocyte transfected with GM-CSF recombinant adenovirus

OBJECTIVE:To compare the biological activities of dendritic cell (DC) derived from monocyte after transfection with adenovirus containing GM-CSF gene with that cultured in cytokine. METHODS: DC was cultured from the peripheral blood monocyte transfected with GM-CSF recombinant adenovirus or by GM-CSF cytokine, and then observed the changes of DC morphology by phasecontrast microscope, analyzed molecules on DC by FACS. Furthermore, the ability to express IL-12 was observed by ELISA, and GM-CSF protein were analyzed by western blot, and the capacity to stimulate the proliferation of allogeneic T cells was examined with MTT. RESULTS: A large number of DC were generated from PBMC transfected with GM-CSF recombinant adenovirus. DC expressed CD1a,HLA-DR,CD80 and CD86 on cell surface, and secreted GM-CSF and IL-12 cytokine. And they were more efficient of inducing allogeneic T cell proliferation in a mixed lymphocyte reaction contrast to control group. CONCLUSIONS: DC may be obtained from the human PBMC modified by GM-CSF adenovirus. This study provides the foundations of DC vaccines for a broadly clinical treatment.