脐血单个核细胞来源红系祖细胞的诱导扩增及保存的研究

The induction and cryopreservation of erythroid progenitor cells derived from umbilical cord bloodmononuclear cells

目的探索含多种细胞因子无血清培养基诱导脐血单个核细胞体外分化为红系祖细胞效率及红系祖细胞的保存方法。方法利用羟乙基淀粉沉降脐血中红细胞，人淋巴细胞分离液（Ficoll）分离单个核细胞，采用含FMS样酪氨酸激酶3配体、干细胞生长因子、胰岛素样生长因子1、重组人红细胞生成素的无血清培养基进行体外诱导脐血单个核细胞向红系祖细胞分化，并将诱导的红系祖细胞用不同的冻存液进行冷冻保存，观察红系祖细胞诱导、分化能力和红系祖细胞冷冻保存效果。结果随着诱导时间延长，细胞总数明显增多，培养14d红系祖细胞扩增约110倍，存活率为（88．92±0．95）％，红系祖细胞特异性标志CD71＋细胞占（86．77±9．11）％，CD71／CD235a细胞占（64．47±16．67）％。诱导10d多为中幼红细胞，可见血红蛋白表达的阳性细胞；诱导14d开始出现晚幼红细胞，细胞沉淀呈红色。诱导7d红系集落数为326．00±97．96，高于诱导前（61．60±20．03）。10％二甲基亚砜（DMSO）＋2％人血清白蛋白保存细胞复苏后红系祖细胞存活率、回收率分别为（90．32±1．80）％、（93．66±1．87）％，将50％自体脐血浆与10％DMSO、2％人血清白蛋白联用可获得更好的保护效果。结论该无血清培养基可高效诱导、扩增红系祖细胞，10％DMSO＋2％人血清白蛋白＋50％自体脐血血浆可很好保存红系祖细胞。

Objective To discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells （EPCs） derived from umbilical cord blood （UCB） mononuclear cells （MNCs）. Methods UCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch （HES）. MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms-liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing. Results With the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14-day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as （88.92±0.95）%. The percentages of cell surface markers were （86.77±9.11 ）% forCD71 and （64.47±16.67）% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells （326.00±97.96 vs 61.60±20.03 per 2 000 ceils）. With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO ＋ 2% HSA showed no different with 10% DMSO ＋ 5% HSA. Combined 50% plasma with 2% HSA was more effective. Conclusions This non-serum culture media could effectively induced and expanded EPCs, and 10% DMSO ＋ 2% HSA ＋ 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.