人脐血CD44^+细胞的分离培养及其分化潜能的初步鉴定

Isolation,culturing and differentiation potentials of CD44^+ cells from human umbilical venous blood

目的分离培养人脐静脉血CD44+细胞,并且诱导其向骨、神经和脂肪组织分化。方法采集的脐血用免疫磁珠阳性筛选法分离CD44+细胞后进行培养。细胞分别行成骨、成神经和成脂诱导分化。观察细胞形态学特征,流式细胞仪检测细胞周期、免疫原性。免疫组化及蛋白印记法检测细胞分化蛋白表达。结果人脐血CD44+细胞贴壁生长,长梭形,传代后细胞增殖迅速。细胞表型为CD44+、CD29+、CD166+、CD34-。在成骨诱导后碱性磷酸酶染色阳性,Von-Kossa染色提示钙化结节形成。成神经诱导后Nestin、NSE、GFAP阳性;Tau和β-tubulin蛋白表达阳性。脂肪诱导后油红O染色阳性。结论脐血中可分离出CD44+细胞,体外诱导可向神经、骨和脂肪方向分化。

Objective To isolate and culture CD44^＋ cells from human umbilical venous blood,and explore their potentials to differentiate into osteogenesis,neurogenesis and adipogenesis in order to select seed cells for tissue engineering.Methods The CD44^＋ cells were isolated by immunomagnetic beads from umbilical venous blood and cultured.The cells at passage 2 were studied with flow cytometry for cell cycle and phenotypes.Then the cells were induced to study the ability of osteogenesis,neurogenesis,and adipogenesis.The cell morphology was observed.The ability of differentiation was tested by immunohistochemiscal assays and Western blot analysis.Results The isolated cord blood CD44^＋ cells were spindle in shape,with the phenotypes of CD44^＋,CD29^＋,CD166^＋ and CD34^-.In osteogenic experiment,the cells were positive to alkaline phosphatase（ALP）,and hydroxyapatite nodules were formed by Von-Kossa staining.Nestin,neuron-specific enolase（NSE）and glial fibrillary acidic protein（GFAP）were positive expressed in neural inducement experiment.Red O staining was positive in adipose differentiation experiment.Conclusion The umbilical venous blood CD44^＋ cells are able to differentiate into osteoid,liparoid and neural-like cells in vitro.