犬骨髓间充质干细胞体外分离、培养后细胞表型变化  

Primary study of cell phenotype of isolated and cultured canine bone marrow mesenchymal stem cells in vitro

作　　者：欧阳辉[1] 刘维永[1] 卫向阳[1] 顾春虎[1] 宿学家[1] 郑奇军[1] 刘兴光[1] 

机构地区：[1]第四军医大学西京医院心血管外科,陕西西安710033

出　　处：《第四军医大学学报》2005年第20期1832-1836,共5页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30271292)

摘　　要：目的:观察犬骨髓间充质干细胞(BMSCs)经分离、自然扩增及诱导分化后表型变化和生物学特性.方法:利用骨髓穿刺、percoll密度梯度法离心分离继而贴壁筛选纯化BMSCs进行接种培养,体外扩增;倒置显微镜下观察其形态学变化,并进行相关抗原检测.结果:分离的细胞免疫组化显示SH2,Vimentin,αSMA和collagenⅠ,Ⅲ均呈阳性表达,其阳性率分别为(95.1±1.7)%,(64.4±2.2)%,(78.7±0.9)%,(78.3±1.4)%和(66.8±0.8)%;CD34,Ⅷ因子antigen和laminin的表达均为阴性.在加入bFGF等诱导培养基作用下,可以诱导分化出成纤维细胞,其诱导分化率为(50.2±2.5)%.诱导后αSMA表达阳性率下降为(42.3±1.6)%,vimentin表达阳性率上升为(86.2±2.4)%,生长扩增能力强,2wk内经3代培养即可扩增到(5.6±0.3)×107细胞数量级.结论:此实验自然分化扩增来的肌纤维母细胞的表型和生物学特征显示这类间质细胞较应用bFGF诱导、分化扩增来的成纤维细胞有更强活力,且分泌胶原力强,更符合构建组织工程种子细胞的要求.AIM. To observe the phenotype changes and biological characteristics of naturally differentiated canine bone marrow mesenchymal stem cells （BMSCs） and differentiated after induced with bFGF in vitro. METHODS： BMSCs were isolated and purified from the canines bone marrow by Percoll density gradient centrifugation and by adhering to the culture plastic. After successive culture and amplification, the growth curve was drawn. The morphology was observed under phase contrast microscope and some antigens were examined by immunohistochemistry stain. RESULTS： SH2, Vimentin, a-SMA, collagen Ⅰ and Ⅲ were all positively expressed in naturally differentiated BMSCs, with the positive percentages （95.1 ± 1. 7） %, （64.4 ±2.2）%, （78.7 ±0.9）%, （78.3 ±1.4）% and （66.8 ± 0.8）%, respectively. CD34, Ⅷ antigen and laminin displayed negative reaction. The induced and differentiated cells showed positive staining of lamininn [ （50. 2 ± 2..5） % ]. The positive expression of α-SMA decreased to （42.3 ±1. 6）% and that of vimentin increased to （86.2±2.4）%. CONCLUSION： The phenotypes and biological characteristics of naturally differentiated myofibroblast with canine BMSCs show that they are more active than those of fibroblasts and more suitable to be used as seed-cells in constructing tissue engineering heart valve （TEHV）.

关 键 词：间质干细胞 骨髓 组织工程 免疫组织化学 犬 

分 类 号：R654.2[医药卫生—外科学]