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目的建立分离培养人脐带间充质干细胞(UC MSCs)和脐带血间充质干细胞(UCB MSCs)的方法,并比较其效果。方法无菌条件下采集健康足月新生儿脐带及脐血各30份,分别通过原代贴壁培养法、酶消化法培养UCMSCs,及淋巴细胞分离液法、羟乙基淀粉沉降与淋巴细胞分离两步分离法获得脐血中单个核细胞培养UCB MSCs。应用含10%胎牛血清的DMEM/F12培养基,比较UC MSCs和UCB MSCs分离培养的效果与生长形态。流式细胞术检测其表面标志及诱导分化鉴定其分化能力。结果 UC MSCs原代贴壁培养法8 d左右可见成纤维样细胞从组织块边缘爬出且成簇生长,酶消化法培养UC MSCs 5 d左右均匀生长;UCB MSCs分离培养用羟乙基淀粉沉降与淋巴细胞分离两步法得到的细胞数量较淋巴细胞分离法明显增多。两种来源MSCs培养方法相比较,UC MSCs原代培养的时间短,培养成功率明显增高。流式细胞仪检测两种来源的MSCs具有MSCs表面标志特征。定向诱导分化结果表明MSCs具有被诱导为成骨细胞、脂肪细胞的分化能力。结论人脐带来源间充质干细胞原代培养周期短,培养效率更高;脐血间充质干细胞的分离方法中羟乙基淀粉沉降与淋巴细胞分离两步法效率更高。
Objective To establish a method to isolate and culture human umbilical cord mesenchymal stem cells (UC MSCs) and umbilical cord blood mesenchymal stem cells (UCB MSCs) and compare their effects. Methods A total of 30 umbilical cord and umbilical cord blood of healthy full-term newborn infants were collected under aseptic conditions. UCMSCs were cultured by primary adherence culture and enzymatic digestion, respectively. Lymphocyte seperation method, hydroxyethyl starch deposition and lymphocyte separation UCB MSCs were cultured with mononuclear cells from umbilical cord blood by two-step separation method. The effect and growth morphology of UC MSCs and UCB MSCs were compared using DMEM / F12 medium containing 10% fetal bovine serum. Flow cytometry was used to detect the surface markers and differentiation to identify their differentiation ability. Results UC MSCs primary adherent culture method about 8 d visible fibroblast-like cells from the edge of the tissue block climbed out and clustered growth, UC MSCs cultured by enzyme digestion uniform growth about 5 d; UCB MSCs isolated and cultured with hydroxyethyl starch sedimentation The number of cells obtained by two-step separation from lymphocytes was significantly higher than that of lymphocyte separation. Compared with the culture methods of MSCs from two sources, UC MSCs primary culture time is short, the success rate of culture was significantly increased. Flow cytometry detection of two types of MSCs with MSCs surface marker characteristics. Directional induction of differentiation results indicate that MSCs have been induced to differentiate into osteoblasts and adipocytes. Conclusion Human umbilical cord-derived mesenchymal stem cells have short initial culture period and higher culture efficiency. The separation of cord blood MSCs is more efficient than the two-step method.