脐带沃顿胶间充质干细胞的分离培养及其诱导分化  

Isolation,culture and differentiation of mesenchymal stem cells from Wharton's jelly of human umbilical cord

作　　者：蒋洁[1,2] 谭灿[1] 张李洋[3] 肖玲[1] 张建湘[1,3] 

机构地区：[1]中南大学湘雅基础医学院组织学与胚胎学系,湖南省长沙市410013 [2]怀化医学高等专科学校组织学与胚胎学教研室,湖南省怀化市418000 [3]中南大学生物科学与技术学院细胞生物学系,湖南省长沙市410013

出　　处：《中国组织工程研究与临床康复》2010年第10期1734-1738,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:骨髓是目前间充质干细胞的主要来源,但由于取材不便、细胞数量受年龄限制等原因,其应用具有一定局限性。近年来,脐带作为间充质干细胞的新来源已经得到广泛关注。目的:探讨从人脐带沃顿胶中分离、扩增间充质干细胞的方法,鉴定其免疫表型与分化潜能。方法:种植法分离和扩增间充质干细胞;用FasGrow培养基培养,光镜观察获得的人脐带沃顿胶间充质干细胞的形态;免疫组织化学方法检测其免疫表型;Gomori钙钴碱性磷酸酶染色、vonKossa钙结节染色、四环素荧光标记鉴定其向成骨方向分化的能力及油红O染色鉴定其向成脂肪分化的能力。结果与结论:种植法容易从人脐带沃顿胶中获得间充质干细胞;原代培养后12～16d达70%～80%融合,传代后可维持未分化状态并稳定增殖;细胞倍增时间为2d左右,体外增殖达20代以上;表面标记分析显示:CD44、CD105、CD133、MHC-Ⅰ呈阳性,CD34、CD45呈阴性;体外诱导实验表明:该细胞具有体外成脂肪、成骨和神经样细胞分化的能力。BACKGROUND： Bone marrow is the main source of mesenchymal stem cells （MSCs） at present, but its application has been limited, because of some reasons such as inconvenience of isolation, and the quantity of cells decreases with human increased age. Umbilical cord as a new source of MSCs has been widespread concerned recently. OBJECTIVE： To explore the approach of isolating and culturing MSCs from Wharton＇s jelly of human umbilical cord, and the methods of identifying the surface antigens and the differentiation potential. METHODS： MSCs were isolated and amplified via tissue-cultivation, and cultured by FasGrow medium. Morphology of MSCs from Wharton＇s jelly of human umbilical cord was observed under the optical microscope. Its immunophenotypes were detected using immunohistochemistry. The differentiation of MSCs into the osteoblasts was determined utilizing Gomori calcium-cobalt alkaline phosphatase staining, von Kossa calcium node staining, and tetracyclinefluorescence labeling. The differentiation of MSCs into the adipocytes was detected using oil red O staining. RESULTS AND CONCLUSION： MSCs were easily obtained from Wharton＇s jelly of human umbilical cord via the proposed approach. The primary cells grew up to 70%-80% confluence after 12-16 days of culture, and meanwhile the undifferentiated state was maintained and proliferation was stabilized after passage. The cell cycle of double increase was about 2 days, and proliferation in vitro reached twenty generation above. Surface antigen analysis showed that CD44, CD105, CD133, MHC-Ⅰwere positively expressed, while CD34, CD45 were negatively expressed. Experiments of differentiation in vitro indicated that the obtained cells were capable of differentiating into fat, osteoblast and nerve-like cells.

关 键 词：脐带 沃顿胶 间充质干细胞 细胞分离 细胞培养 诱导分化 

分 类 号：R394.2[医药卫生—医学遗传学]