梅山猪骨髓间充质干细胞的分离培养及生物学特性分析  

Isolation,cultivation and biological characteristics of bone mesenchymal stem cells in Meishan swines

作　　者：樊懿萱[1] 张艳丽[1] 杨岳[1] 王子玉[1] 宋辉[1] 闫益波[1] 钟部帅[1] 孟立[1] 聂海涛[1] 葛婷[1] 王锋[1] 

机构地区：[1]南京农业大学动物胚胎工程技术中心,江苏南京210095

出　　处：《南京农业大学学报》2011年第6期115-120,共6页Journal of Nanjing Agricultural University

基　　金：国家863计划项目(2008AA101005);国家自然科学基金项目(30771554);南京农业大学青年科技创新基金项目(KJ2010012)

摘　　要：为建立梅山猪骨髓间充质干细胞(BMSC)体外分离培养方法及对梅山猪BMSC的生物学特性进行分析,本研究采用全骨髓法分离培养并传代梅山猪BMSC,倒置相差显微镜下观察细胞形态,以计数法检测细胞增殖状况,流式细胞术检测细胞周期,细胞压片进行染色体分析,免疫细胞化学和RT-PCR法检测细胞表面标志和转录因子的表达情况,并检测成骨和成脂分化能力。结果显示:梅山猪BMSC体外生长良好,细胞形态呈成纤维细胞样,具有稳定分裂增殖能力;CD29、CD44、CD90等表面标志基因及Fou5F1、Sox2、Nanog等转录因子基因表达呈阳性,CD45、Lin28表达呈阴性;在特定诱导液作用下,BMSC可分化为成骨样细胞及成脂肪样细胞。采用全骨髓法成功分离培养出大量纯度较高的梅山猪BMSC,其生长状态良好,可作为后续BMSC相关研究的种子细胞。To develop the methods of isolating and culturing the Meishan swine bone mesenchymal stem cells（BMSC）in vitro,and to investigate their biological characteristics.The Meishan swine BMSC were isolated from the whole bone marrow and cultured,and their morphology was observed by an invert microscope.The proliferation of BMSC was detected by cell counting method,and the growth curve was plotted.Chromosome analysis was checked.The cell cycle,surface markers and pluripotency genes of BMSC in the third passage were analyzed.The directional differentiation of BMSC into osteoblasts and fat like cells were induced,and the differentiation capacity was identified.The results showed that the swine BMSC grew well in vitro,the adherent cells proliferated obviously and most of which were presented in spindle shape on the 5th day.Using specified inducers,BMSC were differentiated to fibroblast-like and fat-like cells.CD44,CD29,CD90,Fou5F1,Sox2,and Nanog could express themselves in BMSC,but CD45 and Lin28 could not.Conclusion：Meishan swine BMSC which was isolated by whole bone marrow method can be purified and well cultured in vitro and maybe served as optimal cell source for further research.

关 键 词：骨髓间充质干细胞 体外分离培养 梅山猪 生物学特性 

分 类 号：S828.81[农业科学—畜牧学] S814.8[农业科学—畜牧兽医]