新生猪骨髓间充质干细胞衍生细胞的分离、培养及生物学特性分析  

Isolation,Culture and Characterization of Derived Cells from Neonatal Porcine Bone Marrow Mesenchymal Stem Cells

作　　者：阮征 王莲芳 胡修忠 吴建英 张四化 代长云 华娟 夏瑜 胡小明[3] 李杰 黄海军 

机构地区：[1]武汉市畜牧兽医科学研究所,武汉430208 [2]武汉市重大动物疫病防控中心,武汉430023 [3]华中农业大学猪遗传育种农业部重点实验室&农业动物遗传育种与繁殖教育部重点实验室,武汉430070 [4]武汉市畜牧兽医局,武汉430023

出　　处：《生物技术通报》2015年第6期170-176,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31201938);武汉市晨光计划(201150431078);武汉市农科院英才计划(YC201101)

摘　　要：利用骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs)治疗疾病已经逐渐成为现实,但是作为被移植的种子细胞,BMSCs体外传代能力非常有限,种子细胞来源极为贫乏。本研究通过差速贴壁筛选的方法分离出一种猪BMSCs的衍生细胞株,命名为猪骨髓间充质干细胞衍生细胞(Bone mesenchymal stem-derived cells,BMSDCs)。分别对BMSDCs与BMSCs细胞进行细胞生物学特性分析,探讨其体外诱导分化特性,并应用流式细胞术测定细胞表面标记物。结果表明,BMSC和BMSDCs细胞倍增时间分别为31.3 h和30.3 h,平均传代时间分别为3-5 d和2-3 d;两种细胞均阳性表达CD34、CD90,阴性表达CD44、CD45;经体外诱导后均可分化为成脂细胞和成肌细胞。在传代能力上,前者可传代15至20次,后者可长期传代(200次以上)且维持正常染色体特征。研究认为在适宜的实验条件下,体外培养的猪骨髓间充质干细胞的衍生细胞——BMSDCs能够稳定生存增殖并维持BMSCs多向分化潜能,可作为组织工程的理想种子细胞。Recently it has become possible to use bone marrow mesenchymal stem cells（BMSCs）in the clinical treatment of certain diseases. However, BMSCs, the seed cells used in transplantation, are limitedin vitro proliferation capability and can only be obtained from very few sources. In order to acquire a substitute for BMSCs, the method of differential velocity adherent screening was used in this study to isolate a derived strain of porcine BMSCs, named bone marrow mesenchymal stem-derived cells（BMSDCs）. The biological characteristics of BMSDCs and BMSCs cells were compared by continuous morphological imaging by inverted microscope, and the growth curves of both cells were monitored by the MTT method. Furthermore, their characteristics of differentiationin vitro were examined, and flow cytometry was used to measure cell surface markers. Our results suggest that the doubling times of BMSCs and BMSDCs are 31. 3 h and 30. 3 h, respectively, and the average passaging time is 3-5 d and 2-3 d, respectively. Cultured BMSCs and BMSDCs are both positive for CD34 and CD90 and negative for＆amp;nbsp;CD44 and CD45. Both BMSCs and BMSDCs can differentiate into adipocytes and myoblasts by induced differentiation in vitro. Concerning their passaging abilities, the former can have 15 to 20 passagesin vitro, whereas latter can have a longer period of time（more than 200 passages） while normal karyotypes are still maintained. We conclude that, under certain experimental circumstances, cultured porcine BMSDCsin vitro can survive and proliferate while maintaining multi-directional differentiation potential of BMSCs, and potentially it can be an ideal type of seed cell for tissue engineering.

关 键 词：骨髓间充质干细胞 衍生细胞 猪 生物学特性 组织工程 

分 类 号：S852.2[农业科学—基础兽医学]