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目的:关于鼠、兔等小动物骨髓间充质干细胞的研究较多,而对大动物骨髓间充质干细胞研究较少。实验拟了解体外培养的羊骨髓间充质干细胞的生物学特性,以便于组织工程研究使用。方法:实验于2006-01/2007-01在天津市天津医院完成。①实验材料:天津医院动物实验中心提供的健康中国山羊,10月龄,实验过程中对动物处置符合动物伦理学标准。②实验方法:抽取羊骨髓5mL,梯度离心制备单个有核细胞,并接种于无菌的50mL塑料培养瓶中,培养体系为含体积分数为0.10FBS的DMEM。利用多孔培养板对培养的骨髓间充质干细胞进行细胞生长曲线测定。选用抗CD44单克隆抗体,应用免疫组化学方法对培养的骨髓间充质干细胞进行表型鉴定。结果:①培养的骨髓间充质干细胞黏附贴壁、呈纺锤状,并有多个突起。②一般细胞接种密度下的骨髓间充质干细胞,三代细胞生长周期基本一致,均为接种培养后前二三天,生长较缓慢,为生长迟滞期。3d后细胞生长加速,有一快速增长过程,达到对数生长期,于七八天时,细胞数达最高值,为生长平台期,其后生长速度减缓。③免疫细胞化学染色后显示所培养细胞的细胞膜抗原CD44有阳性表达。结论:培养的骨髓间充质干细胞具有较强的遗传稳定性和增殖能力,培养的细胞不是骨髓造血干/祖细胞,而是一群均一的间充质干细胞,具有独特的增殖和表型特征。
OBJECTIVE: There are many researches on the bone marrow mesenchymal stem cells of small animals such as rats and rabbits, but few studies on the large animal bone marrow mesenchymal stem cells. Experiment to understand the biological characteristics of cultured sheep bone marrow mesenchymal stem cells in order to facilitate the use of tissue engineering research. Methods: The experiment was performed in Tianjin Hospital of Tianjin from January 2006 to January 2007. ① Experimental material: Healthy Chinese goat provided by Tianjin Hospital Animal Experiment Center, 10 months old, the animals were treated in accordance with animal ethics standards during the experiment. ② Experimental method: 5mL sheep bone marrow was extracted and single nucleated cells were prepared by gradient centrifugation and inoculated into sterile 50mL plastic culture flasks. The culture system consisted of DMEM containing 0.10 FBS. The culture of bone marrow-derived mesenchymal stem cells was subjected to cell growth curve determination using a multi-well culture plate. Anti-CD44 monoclonal antibody was used to detect the phenotype of cultured BMSCs by immunohistochemical method. Results: ① BMSCs adherent adherent wall, spindle-shaped, and multiple protrusions. (2) BMSCs cultured in common cell seeding density showed basically the same growth cycle of three generations, all of them were grown two or three days before inoculation and grew slowly, which was the growth retardation period. After 3d, cell growth accelerated, there is a rapid growth process, reaching the logarithmic growth phase, at seven or eight days, the highest number of cells, for the growth of the plateau, followed by growth slowed down. ③ Immunocytochemical staining showed that the cell membrane antigen CD44 positive expression. CONCLUSION: BMSCs have strong genetic stability and proliferation ability. The cultured cells are not myeloid hematopoietic stem / progenitor cells but a group of uniform mesenchymal stem cells with unique proliferation and phenotypic characteristics.