肿瘤患者造血干细胞来源的树突状细胞的制备及鉴定

目的:以经动员富集的肿瘤患者外周血单个核细胞(MNC)为来源,建立体外诱导培养临床级树突状细胞(DC)的方法并其形态、表型及功能的鉴定。方法:以化疗联合皮下注射G-CSF对肿瘤患者进行外周血干细胞动员,血细胞分离机分离富集外周血MNC,聚苯乙烯细胞培养板贴壁纯化后,加入无血清培养液X-vivo15,以GM-CSF和IL-4联合诱导DC分化,诱导d5加入TNF-α促进DC成熟,观察DC形态及表型变化;并检测其刺激同种异体淋巴细胞增殖的能力。结果:以该法诱导的患者DC显示出典型的树突细胞形态表型特征,同时具有较强的刺激同种异体淋巴细胞增殖的能力。结论:以经动员富集的肿瘤患者外周血MNC为来源,联合应用GM-CSF、IL-4及TNF-α,在体外可诱导培养出临床级具有典型形态、表型及免疫活性的DC。

Generation and identification of dendritic cells from hematopoietic stem cells in patients with solid tumors

Objective： To establish a method to generate dendritic cells （DC） from leukapheresis products （mobilized peripheral blood mononuclear cells）of patients with solid tumor in vitro and to identify the phenotypes and the biological functions of DC. Methods ：After the pretreatment to patients with solid tumor for stem cell mobilization ,leukapheresis products were obtained by cell separator. The obtained cells （mono-nuclear cell enrichment）were purified by density gradient and polystyrene adhesion. They were then cultured in vitro in cytokine-enriched serum-free medium X-vivo 15. On d5 of culture,cells were washed and suspended in medium containing TNF-~ to generate mature DC, which was identified by morphological features, surface antigens expression（analyzed by FCM）. Results：After induction,the cultured cells displayed typical morphology with elongated dendritic processes and had the ability to stimulate high proliferation of allo-T cells. Conclusions： Clinical grade of mature DC could be generated from enriched MNC of patients with solid tumor,which presents the feasibility for further clinical application in the immunotherapy of cancer.