深低温冷冻保存对脐血细胞的影响及机制

Effect of cryopreservation on umbilical blood cells and its mechanism

目的:判断深低温冷冻保存对于脐血单个核细胞和CD34+细胞集落形成能力及凋亡率的影响,从而选取能准确反映冷冻损伤的指标以及优化脐血冷冻保存的方式。方法:从脐血中分离出单个核细胞及CD34+细胞,对这两种形式的细胞进行深低温冷冻保存,保存30 d后复温,并分别进行冷冻前后的粒-巨噬细胞集落形成单位(colony formingunit-granulocyte/monocyte,CFU-GM)和髓系多向造血干细胞(colony formingunit-granulocyte,erythrocyte,monocyte and megakaryocyte,CFU-GEMM)的培养,比较冷冻前后的细胞集落产率,同时AnnexinV–FITC-PI染色后流式细胞仪检测两种保存形式的细胞冷冻前后的细胞凋亡率。结果:冷冻保存对于以单个核细胞形式保存的细胞其集落形成能力和以CD34+细胞形式保存细胞的CFU-GM产率均无明显影响(P=0.31),而对以CD34+细胞形式保存的细胞其CFU-GEMM的产率却有明显影响,冷冻后的CFU-GEMM的产率明显低于冷冻前(P<0.05),且冷冻前即发现两种形式的细胞均有凋亡,冷冻后以单个核细胞形式保存的细胞其细胞凋亡率稍有所上升,但以CD34+细胞形式保存的细胞其细胞凋亡率更高。结论:冷冻前细胞即存在凋亡,冷冻对于以CD34+细胞形式冷冻保存的细胞中的较早期的祖细胞CFU-GEMM存在一定的损伤,其损伤可能主要为诱导细胞凋亡所致,而对于以单个核细胞形式保存的细胞的影响较小。

Objective： To evaluate the effect of cryopreservation on clonogenic ability and apoptosis rate of mono-nuclear cells and CD34＋ cells in umbilical blood （UB）, and to choose the index to present the freezing injury and optimize the cryopreservation of UB. Methods： The mono-nuclear cells （MNC） and CD34＋ cells were separated from UB and frozen.After 30 days, they were thawed in warm water. Clonogenic capacity and clonogenic recovery before and after the cryopreservation was compared. We also used Annexin V-FITC-PI to investigate the apoptosis rate of the cells before and after the cryopreservation of these 2 types of cells. Results： The number of colony forming unit-granulocyte/monocyte （CFU-GMs） was not changed after freezing and thawing in both MNCs and CD34＋ cells, while the number of colony forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte （CFU-GEMM） was obviously reduced after freezing in CD34＋ cells. The 2 types of cryopreserved cells had certain degree of apoptosis before the cryopreservation. MNC-type cryopreservation increased the cells apoptosis a little, while CD34＋-type cryopreservation increased more. Conclusion： The cells have certain degree of apoptosis before the cryopreservation. The freezing and thawing procedure does affect the early stage progenitor cells--CFU-GEMM in the CD34＋- type cryopreserved cells in UB.The damage may be induced by the cell apoptosis.