脐血间充质干细胞肌源性诱导分化的研究

Induction of Mesenchymal Stem Cells from Umbilical Cord Blood into Myoctye in vitro

目的研究脐血间充质干细胞 (MSC)体外的采集、培养和扩增 ,探讨MSC肌源性分化的条件和能力。方法选择杂种妊娠犬 11只 ,采集适量脐带血 ,常规分离后获得单核细胞 ,分别接种于不同的培养液中进行培养和体外传代扩增。免疫组织化学检测脐血MSC表面抗原的表达 ;5 -氮杂胞嘧啶核苷 (5 aza)诱导处理脐血MSC ,观察细胞形态结构的变化及肌源性标记物的表达。结果犬脐血MSC在IMDM中生长良好 ,增殖迅速 ,每传一代细胞扩增 2 ～3倍 ,传至 7代后细胞可扩增 1.5× 10 2～2 .0× 10 3 倍 ;脐血中包含破骨细胞样和纤维母细胞样贴壁细胞 ,后者CD2 9和CD71表达阳性 ,CD11a、CD11b和CD34表达阴性 ;脐血MSC在 10μmol/L的 5 aza诱导 3周后体积延长 ,与邻近细胞形成连接 ,并表达肌节性肌动蛋白和结蛋白 ,与阳性对照结果相似。 结论脐血MSC是存在于脐血中的一类不同于造血干细胞的原始细胞 ,在体外可大量扩增 。

Objective To study the collection,culture and proliferation in vitro of mesenchymal stem cells (MSC) from canine umbilical cord blood (UCB), and to explore the possibility of differentiation of MSC into myocytes. Methods A certain amount of cord blood (5～20 mL) was collected from the hybrid pregnant canine. The mononuclear cells isolated from UCB were seeded in four different culture media: IMDM、a-MEM、DMEM and EBM-Ⅱ. The expression of surface antigens of UCB-derived MSC was detected. The MSC were treated with 5-azacytidine. After observation for 3～4 weeks continuously, then the expressions of several specific antigens of myocytes were detected. Results The canine MSC cultured in IMDM medium expanded rapidly; the cells increased two to three times in number with each passage. The cord blood could give rise to two different kinds of cells, one was the osteoclast-like, and the other fibroblast-like, and the latter expressed antigens CD29 and CD71, but did not express CD11a、CD11b and CD34. The UCB-derived MSC after being treated with 5-aza at 10 μmol/L, were elongated and connected with adjacent cells, and expressed antigen α-actin and desmin, similar to positive control group. Conclusion The UCB-derived MSC are different from the hematopoietic stem cells, and can expand largely in vitro and may differentiate into myocytes under certain circumstances.