人脐血间充质干细胞体外分离、培养与鉴定

Isolation and identification of mesenchymal stem cells from human umbilical cord blood

目的建立体外分离和培养人脐血间充质干细胞的方法,并探讨脐血间充质干细胞是否具有与骨髓间充质干细胞相一致的免疫表型。方法无菌条件下采集正常产胎儿脐血,用相对密度为1.077的淋巴细胞分离液分离脐血间充质干细胞(hu-man umbilical cord blood mesenchymal stem cells,UCB-MSCs),采用贴壁培养法获得MSCs,体外培养并对其增殖进行观察。用流式细胞仪检测细胞周期状态和白细胞表面抗原。结果来源于脐血的MSCs在含有15%胎牛血清和低糖DMEM培养基中可以贴壁,并表现为间充质样细胞;传3代后,此类细胞可以纯化、扩增;其细胞倍增时间为60h左右。流式细胞分析细胞周期显示大部分细胞(85%)处于G0/G1期;此类细胞稳定地表达相关的抗原标记CD29、CD44、CD54、CD105,但不表达造血细胞系的表面标记CD34、CD45。结论脐血中可培养出非造血干细胞,其白细胞表面抗原表达与骨髓间充质干细胞有较强的一致性。

Objective To establish the method of isolation and culture of mesenchymal stem cells（MSCs） from human umbilical cord blood（UCB） and to investigate whether its immunophenotype is coincidence with bone marrow-derived MSCs. Methods Human UCB sample was harvested from full term delivery by cesarean section. The mononuclear cells were separated from UCB using lymphocyted separation medium and suspended ones were removed in the later culture. The adherent cells＇ proliferation and cell cycle were observed and their immunophenotyes were analyzed by flow cytometry（FCM）. Results In low-glucose DMEM, containing fetal bovine serum, adherent cells exhibited mesenchymal-like phenotype. These cells were able to be purified and expanded by three passage. The population-doubling time of cells was approximately 60h. FCM showed that 85 % of cells were in the phase of G0/G1 and these ceils expressed CD29,CD44,CD54 and CD105, but no hematopoietic lineage markers, such as CD34 and CD45. Conclusion Non-hematopoietic stem cells can be isolated in UCB and they have the same immunophenotype with bone marrow-de- rived MSCs.