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背景:内皮细胞基础培养液主要用于培养内皮祖细胞,作者所查用此液培养间充质干细胞的报道较少。目的:对以内皮细胞基础培养液为基础的不同方法培养脐血间充质干细胞的效果比较。设计、时间和单位:对比观察的细胞培养实验,于2005-09/2006-05在上海市新华医院市级重点实验室完成。材料:选取妊娠杂交犬8只,抽取脐血进行干细胞的分离和培养。内皮细胞基础培养液及内皮细胞培养基为美国Clonetics产品。鼠抗CD11a单克隆抗体,鼠抗CD11b单克隆抗体,鼠抗CD29单克隆抗体,鼠抗CD71单克隆抗体均为美国Antibodydiagnostica产品;鼠抗CD34单克隆抗体为美国LabVisionCorporation产品。方法:收集的脐血干细胞分为A,B,C,D4组。A组用内皮细胞基础培养液培养;B组用添加有微血管内皮细胞生长培养液的内皮细胞基础培养液在6孔板上培养。C组用添加有内皮细胞培养基的内皮细胞基础培养液在纤维连接蛋白包被的6孔板上培养。D组用添加有内皮细胞培养基的内皮细胞基础培养液在25cm2细胞培养瓶中培养。主要观察指标:观察各组细胞形态学变化和群体倍增数。应用免疫组化法检测细胞抗原CD11a、CD11b、CD34、CD29及CD71的表达。结果:各组均有成纤维细胞样细胞培养出。A组细胞形态不良,增殖缓慢;B组细胞增殖旺盛;C组细胞克隆不稳定,容易老化;同时出现另一种细胞克隆。D组细胞培养的结果与C组相似。免疫组化法检测抗原显示CD11a(-),CD11b(-),CD34(-),CD29(+)及CD71(+)。结论:在未经处理的6孔板上,用添加有内皮细胞培养基的内皮细胞基础培养液细胞培养液可以培养出生长良好、增殖旺盛的间充质干细胞。应用纤维连接蛋白和25cm2细胞培养瓶培养的效果不佳。
BACKGROUND: The basic culture medium of endothelial cells is mainly used for culture of endothelial progenitor cells. There are few reports about the culture of mesenchymal stem cells by this liquid. OBJECTIVE: To compare the effects of different methods of culturing umbilical cord blood mesenchymal stem cells on the basis of endothelial cell basal medium. DESIGN, TIME AND UNITS: Comparative Cell Culture Experiments were performed at the municipal key laboratory of Xinhua Hospital, Shanghai from September 2005 to May 2006. MATERIALS: Eight pregnant dogs were selected and cord blood was collected for stem cell isolation and culture. Endothelial cell culture medium and endothelial cell culture medium for the United States Clonetics products. Mouse anti-CD11a monoclonal antibody, murine anti-CD11b monoclonal antibody, murine anti-CD29 monoclonal antibody, and murine anti-CD71 monoclonal antibody were both Antibodydiagnostica products from USA; mouse anti-CD34 monoclonal antibody was from Lab Vision Corporation. Methods: Cord blood stem cells collected were divided into A, B, C and D4 groups. Group A was cultured in basal medium of endothelial cells; Group B was cultured in 6-well plates with basal medium of endothelial cells supplemented with microvascular endothelial cell growth medium. Group C was cultured on fibronectin coated 6-well plates with endothelial cell basal medium supplemented with endothelial cell culture medium. Group D was cultured in endothelial cell basal medium supplemented with endothelial cell culture medium in a 25 cm2 cell culture flask. MAIN OUTCOME MEASURES: Morphological changes and population doublings of each group were observed. The expression of CD11a, CD11b, CD34, CD29 and CD71 were detected by immunohistochemistry. Results: Fibroblast-like cells were cultured in all groups. In group A, cell morphology was poor and proliferated slowly; cell proliferation in group B was vigorous; cell clones in group C were unstable and easily aged; and at the same time, another cell clone was found. The results of cell culture in group D were similar to those in group C. Immunohistochemical detection of antigen revealed CD11a (-), CD11b (-), CD34 (-), CD29 (+) and CD71 (+). CONCLUSION: Mesenchymal stem cells, which grow well and proliferate well, can be cultured in endothelial cell basal culture medium supplemented with endothelial cell culture medium on untreated 6-well plates. Fibronectin and 25cm2 cell culture flasks are less effective.