转染融合基因DC抗肿瘤免疫效应.docVIP

转染融合基因DC抗肿瘤免疫效应 　 作者：张文彬 张海红 孔维 查晓 陈廷清 邓碧芳 任原 黄建鸣 【摘要】 目的研究转染融合基因（GMCSFsurvivin GMΔSur）的树突状细胞(DC)在体外诱导高效而特异的抗肿瘤免疫效应。方法用jetPEITMMacrophage转染体系，将构建的GMΔSur融合基因转染入DC，用流式细胞仪检测DC的表面分子HLADR、CD83、CD80、CD86 表达的高低;用LDH法测定转染融合基因的DC诱导特异性细胞毒性T淋巴细胞(CTLs)杀伤肿瘤细胞（HT29和OVCAR3）的能力。结果转染融合基因的DC细胞中可检测到GMΔSur融合蛋白的表达；DC表面高表达HLADR、CD83、CD80、CD86；PHA/rhIL2长期培养（21d）的T细胞CD8+比例明显增加；转染融合基因的DC对HT29肿瘤细胞的杀伤率显著高于未修饰的DC的杀伤率。结论GMΔSur基因转染修饰的DC能选择性诱导MHCI类分子限制的CTL的特异性，显著提高DC的抗原提呈功能和诱导高效而特异的抗肿瘤免疫效应。 【关键词】 肿瘤; 树突状细胞; 融合基因; CTL；survivin； 　　Abstract：Objective To study the specific antitumor immunological effects induced by dendritic cells transfected with GMCSF gene and survivin gene(GMΔSur) in vitro. MethodspcDNA 3.1 plasmid containing GMΔSur fusion gene was constructed and was transfected into dendritic cells by jetPEITMMcrophage transfected kit; cellular surface phenotype such as CD1a, CD80, CD86, CD83 were detected by FACS; The specific cytotoxicity of induced human cytotoxic T lymphocyte (CTLs) to tumor cells was detected with LDH assay. ResultsGMΔSur expression was detected in DC cells; Highlevel expression of HLADR, CD1a, CD80, CD86, CD83 were found in transeneed DC cells; The ratio of CD8+ cells cultured with PHA/rhIL2 were largely increased. The lyse rate of induced CTL cells to HT29 cell were much higher than those of blank DC cells.ConclusionGMΔSur gene modified DC cells could induce MHCⅠspecific cytotoxic T lymphocytes and strikingly raised DC cell,s antigen present function and could induce high effective and specific anticancer immunological effect. 　　Key words：Tumor；Dendritic cell; Fusion Gene; CTL; survivin 　　0引言 　　survivin基因是一种新的抗凋亡基因， 广泛表达于人的胚胎发育组织,在正常成人组织中不表达， 但在大多数肿瘤组织中表达,对肿瘤的发生、 发展起着重要作用， 是一个较为特异的肿瘤相关抗原（Tumorassociated antigen， TAA）基因[1]。 树突状细胞（Dendritic cell，DC）是体内重要的专职抗原提呈细胞， 直接启动和调控机体特异性的抗肿瘤免疫应答[2]。 粒细胞巨噬细胞集落刺激因子（GMCSF）对DC的增殖分化、 细胞活力以及抗原提呈功能有着重要的调节作用[3]。 我们构建的GMCSFsurvivin融合基因表达质粒（pcGMΔSur）并转染人外周血来源诱导扩增的树突状细胞， 探讨转染后DC的生物学特性及诱导产生的抗肿瘤免疫效应。 　　1材料与方法 　　1.