低温冻存脐血树突状细胞的生物特性研究

Study of Biological Properties of Cryopreserved Dendritic Cells Derived from Cord Blood

目的 考察经低温保存的脐血树突状细胞所表现的生物特性 ,为树突状细胞的应用提供保存方法。方法 取新鲜脐血细胞和脐血细胞培养产生的树突状细胞 ,加 5 %二甲基亚砜 6 %羟乙基淀粉的保存剂 ,- 80℃冰箱降温 ,- 196℃保存 ,4 0℃复温 (前者称“冻存脐血” ,后者称“树突状细胞”) ,再用树状突细胞培养增殖法培养 ,检测 :细胞增殖量 ,台盼蓝拒染回收率 ,不染色和染色观察 ,表面抗原 ,树状突细胞的混合淋巴细胞刺激指数、抗毒 杀灭效应处理率 ,比较其变化。结果 形成树突状细胞的时间冻存脐血培养比新鲜脐血迟 ,细胞增殖量低 ,冻存脐血台盼蓝拒染为 95 8%。冻存树突状细胞形成贴壁树突样细胞比新鲜树突状细胞迟 ,细胞数增殖量低 ,冻存树突状细胞的台盼蓝拒染为 88 7%。冻存脐血与新鲜脐血比 ,冻存树突状细胞与新鲜树突状细胞比 ,其树突样细胞量、CD1a、CD83、HLA DR表达低 ;冻存脐血与新鲜脐血比 ,冻存树突状细胞与新鲜树突状细胞比 ,刺激指数、效应处理率均低。结论 用 5 %二甲基亚砜 6 %羟乙基淀粉低温保存脐血细胞或经培养增殖的脐血树突状细胞 ,复温后培养 ,树突状细胞的生物特性受到了损伤 ,但仍完整 ,回收率 >85 %。

Objective To investigate the biological properties of cryopreserved dendritic cells (DCs) derived from cord blood (CB) and to provide a preservative method of DCs for clinical use. Methods Fresh cord blood mononuclear cells (CBMNCs) and DCs produced from cultured CBMNCs (fresh DCs) plus 5%DMSO 6%HES cryopreservatives were frozen in -80℃ refrigerator, then cryopreserved in -196℃ liquid nitrogen. Cryopreserved CBMNCs and DCs thawing in 40℃ proliferated and differentiated in culture into DCs. Morphological changes of DCs in unstained state, increase in cell number and TBRR were observed. DCs were analysed by percentages, expression of surface antigens and functions consisted of stimulating index (SI) measured by MTT method in mixed lymphocyte culture and cytotoxic elminating rate (ER). The results in fresh cells group were compared with that in cryopreserved cells group. Results Cryopreserved CBMNCs differentiated into DCs later and proliferated less than fresh CBMNCs. TBRR was 95.8% cryopreserved DCs became adherent DCs later and grew less in number than fresh DCs. TBRR was 88.7%. After culture the percentages of DCs, expression rates of CD1a, CD83 and HLA DR on the surface were lower in cryopreserved CBMNCs and DCs groups than that in fresh CBMINCs and DCs groups. SI and ER were of the same results. Conclusion Biological properties of CBMNCs and DCs were injured, but basically complete after 5%DMSO 6%HES cryopreservatives. Recovery>85%