大鼠脐带源间充质干细胞的分离及生物学性状**☆

背景：关于大鼠骨髓来源的间充质干细胞用于移植免疫耐受及进行组织修复的研究很多，但尚无脐带来源间充质干细胞的相关研究。目的：建立从大鼠脐带分离间充质干细胞的方法，并观察其生物学性状。方法：大鼠脐带经酶消化和组织块培养两种方法进行分离培养，于DMEM-LG培养基中培养，倒置显微镜观察细胞形态，细胞计数绘制生长曲线，流式细胞仪测定细胞周期及细胞表型，免疫组织化学染色检测其体外诱导成脂肪和成骨分化的能力。结果与结论：两种方法均能成功地从大鼠脐带中获得大量的间充质干细胞：原代培养显示，胶原酶消化法比组织块培养法的效率更高，大约10 d就可以进行传代，而组织块培养法要14 d才能传代；传代扩增两者之间没有差别。免疫表型分析显示，大鼠脐带源细胞表达黏附分子和基质细胞标记CD90、CD106，不表达造血细胞标记CD34、CD45。体外诱导实验证实，大鼠脐带间充质干细胞具有成脂肪和成骨分化的能力。

Isolation and biological characteristics of rat umbilical cord mesenchymal stem cells

BACKGROUND: There are many studies concerning rat bone marrow mesenchymal stem cells for immune tolerance following transplantation and tissue repair. However, there are no reports on umbilical cord mesenchymal stem cells (UCMSCs). OBJECTIVE: To establish a method of separating mesenchymal stem cells (MSCs) from rat umbilical cord, and to study its biological characteristics. METHODS: MSCs were separated from rat umbilical cord with enzyme method and tissue mass method, and then incubated in DMEM-LG medium. Cell morphology was observed under an inverted microscope. Growth curves of cells were drawn using cell counting. Cell cycle and surface antigen were detected with flow cytometry. Adipogenic differentiation and osteogenic differentiation were tested by immunohistochemistry. RESULTS AND CONCLUSION: Both of the two methods could obtain plenty of MSCs from rat umbilical cord. Primary culture showed that the efficiency of enzyme method was higher than tissue mass method. Passage time of the former was about 10 days and the latter was 14 days. The passage time of latter except primary culture was the same. Immunophenotype analysis showed that MSCs from rat umbilical cord expressed adhesion molecule and stromal cell markers, CD90 and CD106, but did not express hematopoietic cell markers, CD34 and CD45. In vitro induction test verified that rat UCMSCs have the potentials of adipogenic and osteogenic differentiation.