人脐血间充质干细胞体外分离培养的实验研究

Isolation and Culture of Mesenchymal Stem Cells from Human Umbilical Cord Blood in Vitro

目的探讨来源于人脐血的间充质干细胞(MSCs)在体外分离、纯化和扩增培养的可行性和条件。方法无菌条件下收集正常足月胎儿的脐带血,经肝素抗凝,用相对密度1.077的淋巴细胞分离液分离脐血的单个核细胞,以偏酸性的M esencu ltTM作为培养基进行培养和纯化扩增,用流式细胞仪检测MSCs的表面标志。结果来源于人脐血的单个核细胞种植于特定的培养基中后,可产生贴壁细胞,主要表现为破骨样细胞和间充质样细胞,传3代后,可得纯化扩增的人脐血MSCs。6.6×105个脐血原代MSCs在体外扩增10代后,获得9.9×108个细胞。流式细胞仪检测结果显示,人脐血MSCs不表达CD34、CD11 a和CD11b,强表达CD29,弱表达CD71,与骨髓MSCs表面抗原特性一致。结论源于人脐血的MSCs在体外可以培养、扩增,可作为干细胞来源之一用于实验研究和临床。

Objective To investigate the feasibility and optimal condition of isolation, purification and expansion of mesenchymal stem cells （MSCs） derived from human umbilical cord blood in vtro. Methods Human umbilical cord blood （HUCB） was collected from full term deliveries scheduled, all samples were obtained sterilely with 20 U/ml preservative free heparin. The cord mononuclear cells were isolated with lymphocyte separation medium （density 1. 077 g/ml）, purified and expanded with MesencuhTM medium and acidic environment to produce adherent layer. The surface antigen expression of MSCs was detected with flow eytometry. Results The HUCB-derived mononuclear cells, when seeded in specific medium, gave rise to adherent ceils, which exhibited either an osteoclast or mesenchymal-like phenotype. After passage 3, these cells were able to be purified and expanded. 6. 6 × 10^5 primary MSCs reached a number of 9. 9 × 10^8 after 10 expanded passage. Flow cytometry showed that MSCs did not express antigens CD34, CD11a and CD11b, but express strongly CD29 and weakly CD71 , which was identical to human bone marrow-derived MSCs. Conclusion MSCs in HUCB can be cultured and expanded in vitro, and could be a source of stem cells for experimental and clinical application.