脐带华通胶间充质干细胞的分离培养及鉴定

Isolation and identification of mesenchymal stem cells from human umbilical cord Wharton's Jelly

目的探讨人脐带华通胶间充质干细胞体外分离、培养方法,并通过成脂成骨诱导及表面标记物的检测进行鉴定。方法剔除脐带动脉、静脉和外膜,取遗留的华通胶组织,将其剪成细小的碎块,用浓度为0.2%的Ⅱ型胶原酶消化,提取脐带华通胶间充质干细胞,流式细胞仪检测细胞表面抗原CD44、CD90、CD105、CD73、HLA-ABC、CD34、CD45及HLA-DR,成骨细胞、成脂细胞分化诱导。结果脐带华通胶经胶原酶消化后,可提取大量间充质干细胞,流式细胞仪检测脐带华通胶干细胞不表达造血干细胞的表面特征,而表达间充质干细胞的标志,例如CD44、CD90、CD105及CD73,并且干细胞HLA-ABC阳性表达,CD34、CD45及HLA-DR呈阴性。组织化学染色显示茜素红、碱性磷酸酶及油红染色强阳性。结论该方法可较好地分离脐带华通胶间充质干细胞,将为实验研究和临床应用提供一种新的干细胞来源。

【Objectives】To explore methods for isolation of mesenchymal stem cells derived from human umbilical cord wharton＇s jelly and to identify the isolated cells which are able to differentiate into osteogenic and adipogenic tissues.【Methods】After removal of umbilical arteries,vein and tunica externa of umbilical cord,the remaining tissue was wharton＇s jelly.To obtain adherent cells,wharton＇s jelly of umbilical cord was cut into pieces and digested with 0.2% collagenase of type Ⅱ.The mesenchymal stem cells of umbilical cord were passaged.Phenotypes were detected with flow cytometry with surface antigens CD44,CD90,CD105,CD73,CD34,CD45,HLA-DR,HLA-ABC and osteogenic and adipogenic tissues were identified by differentiation.【Results】Wharton＇s jelly of human umbilical cord had abundant stem cells.The isolation of these cells had been proved easily when collagenase was used to detach the cells from Wharton＇s jelly.Flow cytometry analyses revealed that the stem cells from Wharton＇s jelly represented a nonhematopoietic cell popution.However they expressed amounts of MSCs markers,such as CD44,CD90,CD105,CD73,meanwhile positive expression of HLA-ABC was found in the stem cells,but not CD34,CD45,HLA-DR.Immunohistochemistry showed that the MSCs strongly expressed alkaline phosphatase and Von Kossa positive.【Conclusions】Mesenchymal stem cells can be successfully isolated from human umbilical cord Wharton＇s jelly.The stem cells derived from Wharton＇s jelly of human umbilical cord may be a novel alternative source of human MSCs for experimental and clinical applications.