脐血CD34＋细胞向内皮细胞诱导分化的体外研究

目的：探讨从脐血中分离CD34＋细胞，并诱导、鉴定其分化为内皮细胞的方法。方法：羟乙基淀粉沉降、密度梯度离心两步法制备脐血单个核细胞，免疫磁珠分选法（MACS）获得CD34＋细胞，在含有细胞因子的M199培养基中诱导。观察细胞形态变化，摄取Dit标记的乙酰化低密度脂蛋白（Dil—AcLDL）的情况，流式鉴定细胞表面标志。结果：分选CD34＋细胞纯度在91％以上，培养3d有明显集落形成及部分细胞贴壁，14d贴壁细胞呈条索状，逐渐增多后呈铺路石样改变。流式检测贴壁细胞表达血管内皮细胞特异性标志物CD144、vWF、CD31、CD34，而白细胞共同抗原CD45为阴性表达，与成熟的脐静脉内皮细胞表达率相近。免疫荧光发现贴壁细胞呈现红色荧光，提示细胞摄取Dil—AcLDL，证明CD34＋细胞分化为有功能的内皮细胞。结论：脐血中存在CD34＋细胞且用两步法和MACS可获得高纯度CD34＋细胞，在特定条件下可分化为内皮细胞。

Induction of human umbilical cord blood-derived CD34+ cells into endothelial cells in vitro

Objective： To study the method of CD34＋ cell isolated from human umbilical cord blood and induced to differentiate into endothelial cells in vitro. Methods： Mononuclear cells were isolated from fresh cord blood by 6 % HES and density gradient centrifugation. CD34＋cells selected from mononuclear cells by magnetic activated cell sorting system （MACS） were cultured in M199 medium supplemented with VEGF, bFGF, IGF-1, then observed the growth and shape of the cells. The cells specific surface mark CD34 and endothelial specific surface marker were assessed by fluorescence activated cell sorter （FACS） analysis. And the cells were also identified by the test of up take of acety-lated low density lipoprotein （Ae-LDL）. Results： The purification of obtained CD34＋cells were more than 95%. Attached spindle-like cells and a number of cell clusters appeared after 3 days culture and cord-like structure by 14 days. When the cells grow into large number, it displayed cobble-stone morphology. These cells express endothelial specific markers, including VE-cadherin, vWF and CD31.The cells＇ function were identified by up take of Ac-LDL. Conclusion： The two methods and the magnetic bead selection are an effective ways to isolate CD34＋ cells from cord blood and there are CD34＋ cells in cord blood, which can differentiate into endothelial cells.