冻融人脐血树突状细胞瘤苗的生物学特征  

Effect of cryopreservation on biological characteristics of cord blood derived DC-based esophageal carcinoma vaccine

作　　者：谢丽华[1] 杨兵[1] 郭光华[2] 俞晶[2] 罗丽莉[3] 

机构地区：[1]深圳市龙岗区人民医院消化内科,深圳518172 [2]汕头大学医学院第一附属医院消化内科 [3]汕头大学医学院第一附属医院妇产科

出　　处：《山西医科大学学报》2008年第7期603-606,共4页Journal of Shanxi Medical University

基　　金：广东省自然科学基金资助项目(021228)

摘　　要：目的利用杂交瘤技术制备脐血树突状细胞和食管癌细胞融合瘤苗,低温冻存,研究冻融后融合瘤苗的生物学特征。方法免疫磁珠法分离脐血CD34+干细胞,多种细胞因子联合诱导扩增为成熟树突状细胞(DC)。聚乙二醇(PEG)融合DC与食管癌细胞EC109,免疫磁珠法筛选阳性克隆EC109-DC为融合瘤苗。-80℃低温冻存;流式细胞术鉴定冻融前后融合瘤苗免疫表型;绘制冻融后融合瘤苗生长曲线;四甲基偶氮唑盐(MTT)法测定EC109-DC融合瘤苗诱导T淋巴细胞体外特异性抗肿瘤免疫应答能力。结果融冻后融合瘤苗EC109-DC可在体外继续培养成长,高表达CD80、CD83、CD86,增殖活性较新鲜融合瘤苗稍低,但两者相比无统计学意义(P>0.05);融冻后融合瘤苗EC109-DC体外能诱导CTL细胞对EC109食管癌细胞的产生特异杀伤作用,其活性与新鲜融合瘤苗相比,差异无统计学意义(P>0.05)。结论融冻后融合瘤苗EC109-DC仍保存其刺激免疫细胞活化的分子表型,可望为食管癌-DC融合瘤苗的保存方法提供一定的实验依据。Objective To study the biological characteristics of cord blood derived dendritic cell（DC）-based esophageal carcinoma vaccine after cryopreservation. Methods CD34 ＋ hematopoietic stem cells were isolated from cord blood using CD34 ＋ prgenitor cell isolation kit and magnetic cell sorting system（MACS）. Then CD34 ＋ cells were expanded to mature DCs with cytokincs rhGM-CSF, rhTNF-α and rhSCF. DCs were fused with EC109 cells by polyethyleneglycol（PEG）. MACS marked with HLA-DR MicroBeads was used to screen the EC109-DC. The fused cells were frozen and stored at - 80℃ for two weeks. After thawing, the immunophenotypes were detected by flow cytometry, and the morphology and the proliferation of the fused ceils were tested by technique of cell culture in vitro. The cytotoxic T lymphocytes（CTL）cytotoxicity response of EC109-DC before and after cryopreservation were examined with MTT assay. Results After cryopreservation, the fused cells still could be cultured in vitro with high expression of CD80,CD83 and CD86,and proliferated slowly in vitro ,but there was no statistic significant difference compared with that of the fresh ones（P 〉 0.05）. The specific CTL activity was well preserved, but compared with the fresh fused vaccine, there was no statistic difference（P 〉0.05）. Conclusion Cryopreservation could not destroy the immunophenotypes of the fused cells. It is promising to be a feasible storage approach for cord blood derived DC-based esophageal carcinoma vaccine.

关 键 词：食管肿瘤 树突细胞 细胞融合 低温保存 

分 类 号：R735.1[医药卫生—肿瘤]