人脐带血清合用胎牛血清培养人脐血间充质干细胞

Human umbilical cord blood mesenchymal stem cells are cultured with human umbilical cord blood serum and fetal bovine serum

目的探讨人脐带血清与胎牛血清合用培养人脐血间充质干细胞(hUCB-MSCs)的形态学、表面标志和胞内标志蛋白的变化。方法密度梯度离心分离脐带血单个核细胞后,应用含10%胎牛血清完全培养基原代培养,分别以3%人脐血清加7%胎牛血清(CBS组)和10%胎牛血清(FBS组)完全培养基传代培养。P5内细胞,进行细胞形态学观察,流式细胞术检测细胞表面分子,免疫细胞化学染色检测细胞内标志蛋白。结果 P5内细胞,两组hUCB-MSCs均为梭型细胞,细胞大小无明显改变,均表达CD44、CD105、CD90、CD73和波形蛋白,不表达CD45、CD34、CD11b、CD19和HLA-DR;其中表达CD44、CD73阳性细胞百分率高达99%。结论 3%人脐血清加7%胎牛血清可成功地培养出形态与表型特征良好的hUCB-MSCs。

Objective To explore the changes of morphology, surface and intracellular marker proteins of human umbilical cord blood mesenchymal stem cells （hUCB -MSCs） cultured with human umbilical cord blood serum and fetal bovine serum. Methods The mononuclear cells were separated by density gra- dient centrifugation from human umbilical cord blood and then primary cultured in complete medium with 10% fetal bovine serum, hUCB - MSCs were subcultured in medium with 10% fetal bovine serum （FBS group） or 3% human cord serum combined with 7% fetal bovine serum （CBS group）, respec- tively, hUCB - MSCs within the 5th generation were prepared for morphological observation. ; examined cells immunophenotype by flow cytometry and immunocytochemical staining were applied for cell immu- nophenotype examination and intracellular marker protein detection, respectively. Results hUCB - MSCs in two groups showed spindle - shaped fibroblasts - liked cells. There was no significant difference of cell size between these two groups. Cells in two groups expressed CD44, CD105, CD90, CD73 and vi- mentin particularly with 99% positive expression rate of CD44 and CD73, whereas the expressions of CD45, CD34, CD11b, CD19 and HLA -DR were negative. Conclusion The morphological and pheno- typic characteristics of hUCB - MSCs could be well cultured with 3% human cord serum t-gether with 7% fetal bovine serum.