脐带源间充质干细胞的分离和生物学性状

Isolation and Biological Characteristics of Mesenchymal Stem Cells from Umbilical Cord

目的建立从脐带分离间充质干细胞的方法,并研究其生物学性状。方法脐带经酶消化后于DMEMLG/F12培养基中培养,倒置显微镜及电镜观察细胞形态学,细胞计数绘制细胞生长曲线,计数成纤维细胞集落形成单位(CFUF),流式细胞仪测定细胞周期及细胞免疫表型,免疫组织化学染色及RTPCR检测其体外诱导成脂肪和成骨分化的能力,RTPCR检测其细胞因子的分泌,与脐血来源CD34+细胞共同培养检测其支持造血的能力。结果经酶消化后,每cm脐带可得到中位数为1.01×106的有核细胞,CFUF产率为1/1609有核细胞,经贴壁传代可分离出成纤维样细胞,细胞倍增时间为(28.02±10.53)h。流式细胞仪分析细胞周期显示,>80%的细胞处于G0/G1期,S+G2+M期的细胞仅占(13.04±4.31)%。免疫表型分析显示,CD13、CD29、CD44、CD105(SH2)、CD73(SH3)、CD166和MHCI阳性,CD45、CD34、CD38、CD31和MHCⅡ阴性。体外诱导实验证实,该细胞具有成脂肪和成骨分化的能力。RTPCR显示,该细胞表达干细胞因子、血小板生成素,酪氨酸激酶受体配基、白细胞介素6、巨噬细胞集落刺激因子、白血病抑制因子、基质细胞衍生因子和血管内皮细胞生长因子,不表达白细胞介素3。与脐血来源CD34+细胞共同培养2周可见鹅卵石形成区,共培养8周证实其支持长期造血能力。结论建立从脐带中分离间充质干细胞的方法,脐带是间充质干细胞的新的来源。

Objective To isolate the mesenchymal stem cells from umbilical cord and identify its biological characteristics. Methods Mesenchymal stem cells（MSC） have been isolated from venous endothelia/subendothelia, Warthon＇s jelly and prevascular tissue of the human umbilical cord（UC） using a established simple method. 36 full-term UC were recruited after informed consent. MSC were isolated after enzyme digestion of minced cord fragments. Results The median of nucleated cells isolated was 1.01× 10^6 per cm-long UC. A total of 1 × 10^10 MSC was obtained in 4 weeks. CFU-F productive was 1 ： 1 609 of nucleated cells. The population doubling time was approximately （28.02±10.53）h. The MSC were positive for CD13, CD29, CD44, SH-2, SH-3, CD166 and MHC-Ⅰ, but were negative for CD34, CD38, CD45, CD31 and MHC-Ⅱ. More than 80% of cells were in G0-G1 phase, whereas a small population of cells was engaged in proliferation （S＋G2 ＋M= 13.04%±4.31%）. Under specified culture conditions, the MSC differentiated into adipocytes and osteoblasts cells. The MSC were also found to express cytokines of SCF, LIF, M-CSF, Flt3-ligand, IL-6, GM-CSF, G-CSF, VEGF and SDF-1. When co-cultured with CD34^＋ cells from cord blood（CB）, the UC-derived MSC were able to support the hematopoiesis of long-term culture-initiating cells. Conclusion These findings suggested that abundant MSC can be isolated simply and effectively from the whole cord tissue. UC may be an attractive source of MSC for tissue engineering, CB expansion and cord blood transplantation.