成人骨髓间充质干细胞体外培养及其生物学特性鉴定

Isolation,culture and biological characteristics of bone marrow-derived mesenchymal stem cells in adult human with combined density gradient centrifugation and attachment culture method

目的探讨并优化成人骨髓间充质干细胞（hBMSCs）体外分离培养方法，鉴定其生物学特性，为BMSCs应用奠定技术基础。方法将手术弃骨无菌条件下用无血清培养液（α-MEM培养基、100u／ml青霉素，100u／ml链霉素）冲出骨髓，采用密度梯度离心法结合贴壁筛选法分离纯化hBMSCs，通过传代培养，扩增hBMSCs。倒置光学显微镜下观察细胞形态及生长情况，细胞计数法绘制细胞生长曲线，并分析冻存细胞复苏后的生长特性，免疫荧光染色测定hBMSCs表面标志，成骨诱导试剂盒检测hBMSCs向成骨分化的潜能。结果倒置显微镜下观察hBMSCs呈梭形、多角形成纤维细胞样形态，大小均一，漩涡状生长。细胞生长曲线分析细胞贴壁2d基本无增殖，处于潜伏期，对数增殖期为3—6d，第7天后进入平台期，细胞出现接触抑制现象。冻存细胞复苏后细胞生长特性无明显改变。免疫荧光染色结果显示，第3代hBMSCs小鼠IgG、CD34、CD45表达阴性；CD29、CD90、CD166表达阳性，均为胞浆着色，细胞表型稳定。成骨诱导试剂诱导细胞10d后hBMSCs表现成骨细胞特性，且碱性磷酸酶活性明显增高。结论密度梯度离心法结合贴壁筛选法分离纯化的hBMSCs纯度高，增殖活性强，传代培养生长旺盛且生物学特性稳定；可为组织细胞工程和基因治疗提供理想的种子细胞。

Objective To study and establish a more effective and appropriate method to isolate and cultivate the human bone thy mal cells（hBMSLs） obtained from adult human bone marrow in vitro and identify the biological properties for further study and clinic application. Methods The marrow which derived from deserted bone after operation was flushed out with free-serum α-MEM medium under aseptic condition, hBMSCs were isolated and purified by density gradient centrifugation combined with attachment culture method. The growth and mor- phology of the primary culture and subculture were observed under inverted phasecontrast microscopy and the growth curve was detected by cell counting. The biological features of hBMSCs and cryogentic storaged cells were analyzed. hBMSCs memberane antigens were identified by immunofluorescence staining. The potential osteogenous differentiation of hBMSCs was evaluated by the osteogeneic inducer kit. Results The observation of growth and morphology of hBM- SCs ：after seeded 48 hours,the adherent cell showed spindle shape, polygonal shape and fibroblast-cell-like shape and the size of hBMSCs was homogeneous. The morphology of subculture was more homogeneous. The cell growth curve of hBMSCs ：The hBMSCs were still in latent phase after being adherent to the bottom for 2 days;3- 6 days later, cells were in log phase;7 days later,and cells came into platform phase and displayed contact inhibition. The biological features of cryogentic storaged cells did not alter. CD29 ,CD90 ,CD166 memberane antigen could be detected in 99% of the third generation of hBMSCs,but not CD34,CD45 memberane antigen,and the phenotype of hBMSCs was stabile. After osteogeneic inducer was added in vitro for ten days, the alkaline phosphatase activities were increased obviously. Conculusions hBMSCs isolated and purified by density gradient centrifugation combined with attachment method has high purity and proliferation activity. The biological properties was stabile. It could provide ideal seed cells for tissue engineering and gene therapy.