冻存对脐血树突状细胞介导的食管癌疫苗的影响

Cryopreservation influence on biological characteristics and CTL cytotoxicity of cord blood derived DC-based esophageal carcinoma vaccine

目的:观察低温冻存对人脐血DC与EC109食管癌细胞融合疫苗生物学特性及CTL活性的影响。方法:应用免疫磁珠标记法分选人脐血CD34+干细胞,细胞因子扩增培养为成熟DC,聚二乙醇(PEG)融合法制备EC109-DC融合疫苗,-80℃低温冰箱冻存,3周后复苏,计算疫苗回收率及存活率。倒置相差显微镜观察冻存后疫苗的细胞形态,流式细胞术分析疫苗及疫苗活化后T淋巴细胞的表型,MTT法检测疫苗特异的CTL活性。结果:冻存后疫苗存活率及回收率分别为(77·2±1·8)%、(61·8±1·4)%。冻存对疫苗细胞形态及表型无明显影响。冻存疫苗和未冻存疫苗活化后T淋巴细胞,CD3+T/CD4+T及CD3+T/CD8+T均较活化前明显增加,P=0·002;CD4+T/CD8+T由活化前的0·85分别增加至1·25和1·29,疫苗冻存与否对T细胞活化能力无明显差别,P=0·052。疫苗特异的CTL活性未受冻存明显影响,其杀伤率与未冻存疫苗组相比差异无统计学意义,P=0·667。结论:-80℃低温冰箱短期冻存对EC109-DC融合疫苗生物学特性及特异的CTL活性无明显影响,可望为EC109-DC疫苗的开发应用提供简单可行的保存方法。

OBJECTIVE： To study the biological characteristics and specific cytotoxic activity of cord blood derived DC-based esophageal carcinoma vaccine after cryopreservation. METHODS： CD34^＋ hematopoietic stem cells were isolated from cord blood by using CD34^＋ progenitor cell isolation kit of magnetic cell sorting system （MACS）. Then the CD34^＋ cells were expanded with cytokines togetmature DCs, and then fused with EC109 cells by PEG-3600. There fused cells were frozen and stored at -80℃ for three weeks. After thawing, the cells were assayed for their viability, morphology, immunophenotypes and T-cell stimulatory capacity. Specific CTL activity was also tested by MTT. RESULTS： Cryopreservation did not cause significant changes in the phenotypes or morphology of the fused cells, and the recovery rate and trypan blue viability were （61.8±1.4）% and （77.2±1.8）% respectively. The numbers of CD3^＋ T/CD4^＋ T and CD3^＋ T/CD8^＋ T lymphocytes increased after being stimulated by DC-based esophageal carcinoma vaccine either before or after cryopreservation, and the ratio of CD4/CD8 increased from 0.85 to 1.29 and 1.25 respectively, and the function of T-cell stimulating capacity and the specific CTL activity were well preserved （compared with the fresh fused vaccine, P = 0.667）. CONCLUSIONS： A simple short-term -80℃ freezing and storage method is practical. It is promising to be a new storage approach for cord blood derived DC-based esophageal carcinoma vaccine during the clinical application.