不同年龄兔骨髓间充质干细胞的体外生长特性分析

In vitro growth of bone marrow mesenchymal stem cells from rabbits at varied ages of life

目的观察不同年龄兔骨髓间充质干细胞(BMSC)在体外分离、培养、扩增的过程,并对其生长特性进行初步比较分析,为BMSC的临床应用提供技术方法和依据。方法选取出生1月、18月、36月的健康中国白兔,分成新生、成年、老年3组,采用密度梯度离心技术及贴壁筛选法相结合,分离培养BMSC,用噻唑蓝(MTT)法观察其增殖及生长特征,用流式细胞仪检测细胞表面标志。结果各组BMSC均贴壁生长,呈成纤维细胞样BMSC典型形态,贴壁率的观察和生长曲线显示,在相同的培养条件下,新生兔细胞增殖最快,细胞可扩增能力最强,而老年兔细胞增殖最慢,细胞可扩增能力最弱,成年兔细胞介于两者之间。结论利用密度梯度离心法结合贴壁培养法可以从不同年龄兔中获得高纯度的BMSC,干细胞的活性和增殖能力随着年龄的增加而降低。

Objective To explore the isolation, culture and passage of marrow mesenchymal stem cells （BMSCs） from rabbits at different ages in vitro, and to investigate the patterns of BMSCs growth. Methods Rabbits aged 1 month （newborn group）, 18 months （adult group） and 36 months （elder group） were used to collect BMSCs. BMSCs were separated and purified with density gradient centrifugation combined with adherence method in vitro. The proliferation and pattern of growth were studied by MTT essay in primary and passage culture. The surface molecule expressions of cells were examined by flow cytometry. Results BMSCs were found to adherent to walls and present with a typical morphology of fibroblasts. The growth curve and adhesive rate showed that cells from newborn rabbits grew faster than those from adult and elder rabbits under the same setting of culture. Conclusion Highly purified BMSCs can be obtained from rabbits with different ages by gradient centrifuge and adherent culture. The BMSCs from younger rabbits showed a stronger capability of growth and proliferation compared with those from the elder ones.