体外培养羊骨髓间充质干细胞增殖及表型特征  

Proliferation and phenotype characteristics of goat bone marrow mesenchymal stem cells cultured in vitro

作　　者：曹希武 许效坤 苗军[2] 王秀芬 寇炳祯 左炳光 谢晨学 丁光宁 曹富余 杨利 

机构地区：[1]沧州市人民医院骨三科,河北省沧州市061000 [2]天津市天津医院脊柱外科,天津市300211

出　　处：《中国组织工程研究与临床康复》2008年第3期465-468,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30500516)~~

摘　　要：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。AIM： Studies on bone marrow mesenchymal stem cells are mostly in small animals such as mice or rabbits, but few in big animals. This study researched the biological features of bone marrow mesenchymal stem cells from goat used in tissue engineering. METHODS： Experiments were conducted at Tianjin Hospital from January 2006 to January 2007. （1）A Chinese goat aged ten month was offered by Animal Experiment Center of Tianjin Hospital. Animal intervention met the animal ethical standard. （2）The mononuclear cells were isolated from 5 mL of fresh bone marrow by gradient centrifugation, and then inoculated in dishes. Cells were cultured and expanded with Dulbecco＇s modified Eagle＇s medium containing 0.10 volume fraction fetal bovine serum. Cell growth curve was determined with multiple-well culture plate. Phenotype of bone marrow mesenchyrnal stem cells was determined by immunohistochemical method with antibody of CD44. RESULTS： （1）Cultured bone marrow mesenchymal stem cells adhered to the wall, appeared morphologically as spindle and had many prominences.（2）Growth cycle of three generations of bone marrow mesenchymal stem cells at common inoculated density was similar. Two or three days before culture was growth lag phase. Three days later, cells grew rapidly and entered logarithmic phase. Seven or eight days later, the amount of cells was maximal and entered growth platform phase, and then cells grew slowly. （3）Immunocytochemical staining showed bone marrow mesenchyrnal stem cells were positive for CD44. CONCLUSION： The cultured bone marrow mesenchymal stem cells are not hematopoietic stem/progenitor cells. Bone marrow mesenchymal stem cells are a homogenous population of cells that have unique growth and phenotype characteristics.

关 键 词：羊 骨髓间充质干细胞 体外培养 鉴定 

分 类 号：R394.2[医药卫生—医学遗传学]