表皮生长因子对非黏附骨髓间充质干细胞克隆形成效率的影响  

Effects of epithelial growth factor on colony-forming efficiency of non-adherent bone marrow mesenchymal stem cells

作　　者：王燕[1] 陈燕玲[2] 贲晓明[2] 苗登顺[2] 

机构地区：[1]泰安市中心医院新生儿科,山东省泰安市271000 [2]南京医科大学附属南京儿童医院新生儿医疗中心,江苏省南京市210008

出　　处：《中国组织工程研究与临床康复》2009年第23期4475-4479,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金项目(30471836);江苏省卫生厅重大项目(2004-04)~~

摘　　要：背景：目前普遍认为骨髓间充质干细胞是一类高度黏附的成纤维样细胞，但也有研究显示处于非黏附状态的骨髓间质细胞也具有自我复制及多向分化潜能。目的：探讨表皮生长因子对骨髓中存在的非黏附骨髓间充质干细胞产生成纤维细胞集落形成单位的影响。设计、时间及地点：细胞学体外观察，于2007-07／12在南京医科大学骨与干细胞研究中心完成。材料：4周龄雄性C57／BL6小鼠6只，由南京医科大学实验动物中心提供。表皮生长因子为美国Sigma公司产品。方法：全骨髓法体外分离培养小鼠骨髓间充质干细胞，调整细胞浓度为2×10^9L^-1。收集第3代细胞，分别接种于向脂肪细胞、成骨细胞、软骨细胞分化的培养液中进行诱导。取30mL骨髓间充质干细胞悬液，平均分到两管中，表皮生长因子处理组加入终浓度为10μg／L表皮生长因子，空白对照组加入普通完全培养基，采用反复转移非黏附骨髓细胞培养法，每天将非黏附骨髓细胞转移到新的培养皿，原来的皿中为总骨髓来源的贴壁细胞，转移第1次定义P01，第2次为P02，依次类推，培养12d终止。主要观察指标：骨髓间充质干细胞的鉴定结果，亚甲基蓝染色显示每皿细胞克隆的形成情况，计算克隆形成区域占皿底面积的百分比及克隆数目。结果：非黏附骨髓间充质细胞在第4天转移到新的培养皿后，继续培养仍能够贴壁生长，并逐渐扩增形成克隆；诱导后能够分化为脂肪细胞、成骨细胞和软骨细胞。给予表皮生长因子处理后，无论是总骨髓来源的贴壁细胞，还是反复转移的非黏附骨髓间充质干细胞，均能够不断产生成纤维细胞集落形成单位，且数量明显多于空白对照组（P〈005）。表皮生长因子处理组总骨髓来源的贴壁细胞、P01-P05非黏附骨髓间充质干细胞所形成的贴壁细胞数分别是空白对照组�BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） are a kind of fibroblast-like cells with high adhesion. Some studies showed that BMSCs in non-adherent status have the potential of self-duplicating and multi-directional differentiation. OBJECTIVE： To explore effects of epithelial growth factor （EGF） on fibroblastic colony forming units （CFU-F） from the non-adherent BMSCs. DESIGN, TIME AND SETTING： The cytological, in vitro study was performed at the Research Center of Bone and Stem cells, Nanjing Medical University from July to December 2007. MATERIALS： Six male C57/BL6 mice aged 4 weeks were supplied by Experimental Animal Center, Nanjing Medical University. Epidermal growth factor （Sigma, USA） was used in this study. METHODS： Mouse BMSCs were isolated in vitro by the whole bone marrow method. The third passage of cells at 2×10^9/L were incubated in adipogenic, osteogenic and chondrogenic mediums, separately. 30 mL BMSC suspension was obtained and averagely assigned into two tubes. Cells in the EGF group were treated with 10 μ g/L EGF. Cells in the blank control group were treated with common complete medium. Using repeat transfer non-adherent bone marrow cell method, non-adherent bone marrow cells were moved in the a new Petri dish every day. Adherent cells in the original dish were derived from the total bone marrow. Cells were named PO1 following the first transfer, and PO2 following the second transfer, and so on. Culture was stopped at day 12. MAIN OUTCOME MEASURES： Determination results of BMSCs; methylene blue staining showed that cell clone formation in each dish; percentage of clone forming region to dish bottom area; clone number. RESULTS： Non-adherent BMSCs could adhere at day 4 following moving into a new dish, and gradually amplify into clones. Following induction, these BMSCs could differentiate into adipocytes, esteoblasts and chondrocytes. Following EGF treatment, both total bone marrow-derived adherent cells and transferred non-adherent BMSCs could p

关 键 词：非黏附骨髓间充质干细胞 表皮生长因子 成纤维细胞集落形成单位 

分 类 号：R394.2[医药卫生—医学遗传学]