人脐静脉间充质干细胞的分离培养及生物学特性鉴定

Isolation and Culture of Human Umbilical Cord Vein Mesenchymal Stem Cells and Identification of Their Biological Characteristics

为了对人脐静脉间充质干细胞(MSC)进行分离培养及其生物学特性鉴定。采用胶原酶分步消化法获得人脐静脉间充质干细胞(hUVMSC2)并对其进行体外培养、形态学观察及绘制生长曲线;利用条件培养基诱导法分析该细胞分别向成骨细胞和脂肪细胞分化能力;流式细胞术检测细胞表面标志物CD54、CD105、CD29、CD166、CD44、CD31、CD34、CD49、CD106等表达情况。结果该细胞形态为梭形或成纤维样,不表达内皮来源的vWF因子。在不同诱导条件下,该细胞可分别向成骨细胞和脂肪细胞分化。hUVMSC2细胞表面表达CD54、CD105、CD29、CD166、CD44等间质细胞黏附分子,不表达CD31、CD34、CD49、CD106等内皮或造血细胞相关标志物。该细胞指数生长期倍增时间约为26h,在添加bFGF条件下可迅速增殖,指数生长期倍增时间缩短为16h。研究证实人脐静脉内皮层下存在间充质干细胞,采用分步酶消化法可同时分别获得单根脐静脉的内皮细胞和间充质干细胞。hUVMSC2间充质干细胞具有向脂肪细胞和成骨细胞分化潜能并表达多种黏附分子。

To isolate and culture human umbilical cord vein mesenchymal stem cells (MSCs) and identify their biological characteristics, sequential collagenase digestions were adopted to isolate human umbilical cord MSCs. Morphology and cell growth curve were detected in hUVMSC2 cell line. Different induction conditions were used to direct hUVMSC2 cells to differentiate into osteoblasts and adipocytes respectively. Flow cytometry was adopted to analyze expression of surface markers such as CD54, CD105, CD29, CD166, CD44, CD31, CD34, CD49, and CD106, etc. Morphologically hUVMSC2 cells were fibroblast-like, did not express endothelial-specific marker vWF. Under different induction conditions, hUVMSC2 cells could be directed to differentiate into osteoblasts and adipocytes respectively in vitro. Through flow cytometry assay, hUVMSC2 cells express adhesion molecules such as CD54, CD105, CD29, CD166, CD44, but not CD31, CD34, CD49, and CD106 endothelial or hematopoietic markers. During exponential growth period, hUVMSC2 cells doubling time is about 26 h, whereas when mitogen bFGF added, cells grew faster and the doubling time became shorter as16 h. This investigation confirmed that human umbilical cord vein mesenchymal stem cells exist under endothelia layer. And sequential collagenase digestion method could successfully be used to isolate and culture two types of cells as endothelial cells and MSCs simultaneously from a single human umbilical cord vein. These MSCs had bi-directional differentiation potential at least, and also expressed important adhesion molecules such as ICAM-1, β3-integrin, and ALCAM, etc. This study also provided new MSC origin and basic data for stem cell study and clinical MSC application in combination with hematopoietic stem cell transplantation.