兔骨髓间充质干细胞的生物学特征及原代培养

背景：兔来源的骨髓间充质干细胞是具有较强的体外增殖和多系统分化潜能的成体干细胞，在组织工程及生物治疗领域蕴藏着巨大的潜能。 目的：体外培养扩增、鉴定兔骨髓间充质干细胞，观察骨髓间充质干细胞的生物学特征。 方法：无菌条件下抽取兔骨髓，分别运用全骨髓贴壁法和Percoll密度梯度离心法体外分离骨髓间充质干细胞，利用差速贴壁原理对细胞进行纯化扩增。在显微镜下观察细胞形态特征及生长规律，流式细胞技术检测细胞表面抗原标记物的表达。 结果与结论：兔骨髓间充质干细胞贴壁时间短，增殖快，经过细胞传代后能够获得进一步纯化的细胞，杂质细胞减少。原代细胞形态即呈现三角形、长梭形、纺锤形的贴壁细胞特征。第 5代骨髓间充质干细胞呈典型的极性漩涡状生长，形态单一均匀，不具有表达造血前体细胞表面标志抗原CD34和白细胞表面标志抗原CD45的表面标记物功能，但是具有能够表达出整合素家族的成员 CD29 及黏附分子CD44的特点。说明经全骨髓贴壁法和Percoll密度梯度离心法体体外分离培养的细胞在形态学、细胞表面标志物表达和多向分化能力方面具有干细胞生物学特性，经流式细胞分析鉴定为兔骨髓间充质干细胞。 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Primary culture and biological characteristics of bone marrow mesenchymal stem cells from a rabbit

BACKGROUND: Rabbit bone marrow mesenchymal stem cells as a kind of adult stem cells with strong proliferation and multilineage differentiation potential exhibit a tremendous application potential in tissue engineering and biological therapy. OBJECTIVE: To in vitro culture, proliferate and identify rabbit bone marrow mesenchymal stem cells and to observe cell biological characteristics. MEHTODS: Bone marrow of rabbits was extracted under sterile conditions to separate bone marrow mesenchymal stem cells using the whole bone marrow adherence method and Percoll density gradient centrifugation method. Afterwards, the cells were purified and proliferated using differential adherence method. Morphology and growth pattern of cells were observed under microscope, and expression of cell surface antigen markers was detected by flow cytometry. RESULTS AND CONCLUSION: Rabbit bone marrow mesenchymal stem cells presented with short adherent time and fast growth. After passage and purification, impurities cell counts were decreased. Primary cells presented with triangular, fusiform and spindly shapes. Passage 5 cells with single shape showed the typical polar swirling growth, and could not express CD34 and CD45, but expressed CD29 and CD44. These findings indicate that the cells cultured using the whole bone marrow adherence method and Percoll density gradient  centrifugation method possess stem cell characteristics in morphology, surface markers and multilineage differentiation, which have been identified as bone marrow mesenchymal stem cells by flow cytometry.