人脐带间充质干细胞的体外分离、培养及诱导分化

Isolation,culturation,and induced differentiation of human umbilical cord mesenchymal stem cells in vitro

目的：体外分离、纯化及培养人脐带间充质干细胞（MSCs），并观察其生物学特性。方法：将剔除动静脉的新鲜人脐带组织切成小块培养，得到贴壁细胞，倒置显微镜观察细胞形态，绘制第1、5及10代细胞生长曲线；流式细胞仪测定细胞表面标记（CD13、CD44、CD14、CD34与HLA—DR）；化学染色（油红O、茜素红染色）及RT—PCR检测其体外诱导成脂和成骨分化的能力；RT—PCR检测胚胎干细胞特异性标志基因OCT-4。结果：体外培养4～6d后，有细胞从组织块中游出；细胞传代培养达10代后无明显的形态和增殖能力改变。培养细胞表达CD13与CD44，但CD14、CD34及HLA—DR呈阴性表达。体外诱导实验证实，该细胞具有成脂和成骨分化的能力。RT—PCR屁示其表达OCT-4基因。结论：人脐带MSCs能在体外培养、扩增并且具有和骨髓MSCs类似的生物学特性，可作为组织工程种子细胞来源。

Aim ： To explore the conditions of isolation, purification,and culture of human umbilical cord mesenchymal stem cells （MSCs） in vitro, and study their biological characteristics. Methods：After stripping off arteries and veins, the re- maining parts of umbilical cord were cut into small sections and cultured. Adhere ceils were obtained, and the morphology of the cells was observed under inverted phase contrast microscope. The growth curves of them were drawn and the surface an- tigens of human umbilical cord MSCs （CD13 ,CD44 ,CDI4, CD34 ,and HLA-DR） were detected by flow cytometry. The po- tentiality of their osteogenic and adipogenic differentiation was detected by chemistry staining and RT-PCR, and the stem cell marker gene OCT-4 was detected by RT-PCR. Results：Four to six days after primary culture,adhere cells came out of fragments and could be maintained for 10 passages without obvious changes in morphology or growth pattern. Flow cytometry analysis revealed that CD13 and CD44 were expressed on these cells＇ surface,while CD14, CD34, and HLA-DR were negative