大鼠骨髓间充质干细胞的分离与培养贴壁分离和消化控制相结合可否为简便高效的技术方法

Cultivation and separation of rat bone marrow-derived mesenchymal stem cells:Is adhesion separation plus digestion control a simple and effective method or not

目的:对骨髓间充质干细胞的培养一般采用流式细胞仪分离法及密度梯度离心法和免疫磁珠分离法,本实验观察了采用贴壁分离和消化控制相结合方法分离纯化的可行性。方法:实验于2004-09/12在锦州医学院附属第一医院外科实验室完成。选取6～8周龄的SD大鼠,雌雄不限,从其股骨、胫骨中取材,在无菌条件下操作,用剪刀分别剪去股骨胫骨的两端,暴露骨髓腔,用含有体积分数为0.15的胎牛血清Dulbecco改良培养基从骨髓腔中冲出骨髓于平皿中,用含有体积分数为0.1的胎牛血清的Dulbecco改良培养基培养,用消化控制及贴壁分离法分离纯化骨髓间充质干细胞,并观察其生长状态,测定骨髓间充质干细胞的生长曲线,分裂指数,贴壁率,用免疫细胞化学方法对骨髓间充质干细胞进行表型分析。结果:原代培养的骨髓间充质干细胞呈椭圆型、短梭型、长梭型、多角型等。纯化、扩增后骨髓间充质干细胞呈均匀一致的长梭型,第6代前生长性状稳定,增殖能力强。传代周期为7d,每次传代时细胞活力测定均>97%,传代后24h内贴壁率99%。免疫细胞化学检测第3代细胞均匀表达CD54(ICAM-1)、纤维连接蛋白。结论:实验建立了一种可行的大鼠骨髓间充质干细胞取材、纯化、扩增方法。培养的骨髓间充质干细胞纯度高,细胞活力强,生物学性状稳定。本实验通过贴壁分离和消化控制相结合的培养方法具有操作简便,条件要求低等优点,是目前一种比较理想的分离纯化方法。

AIM:Flow cytometry separation,density gradient centrifugation and immunomagne tic beads separation are commonly employed for the culture of bone marrow- deri ved mesenchymal stem cells(MSCs).This study aimed to investigate the feasibility of adhesion separation plus digestion control for the culture of MSCs from rat bone marrow. METHODS:The experiment was completed at surgical laboratory of the First Affi liated Hospital of Jinzhou Medical College from September to December 2004.MSCs were isolated from the thighbone and shankbone of SD rats in either gender of 6 to 8 weeks.Under aseptic condition,two ends of thighbone and shankbone were both sheared with a scissor to expose the bone medullary cavity,from which the bone marrow was rinsed with Dulbecco's modified Eagle's medium(DMEM) containing 150 m L/L fetal bovine serum onto a plat,and then the bone marrow was cultured in DMEM containing 100 mL/L fetal bovine serum.They were separated and purified by dige stion control and adhesion separation followed by the observation of growth stat us.The growth curve of MSCs was drawn,cell division index and adhesion rate were assessed,and the phenotype of MSCs was analyzed by immunocytochemistry. RESULTS:MSCs cultured primarily were oval,short spindle- shaped,long spindle - shaped or polygonal.After purification and proliferation,they were uniformly long spindle- shaped form.The growth behavior of MSCs from passage 2 to passage 6 was quite stable,exhibiting a large proliferative potential.MSCs were passage d every seven days.The cell activity was assayed more than 97% every passage,a nd the adhesion rate within 24 hours was 99% .MSCs of passage 3 uniformly expre ssed CD54(ICAM- 1) and fibronectin by immunocytochemistry. CONCLUSION:A feasible method for the separation,purification and proliferatio n of MSCs from rat bone marrow has been established.The cultured MSCs are active in high purity with stable biological character.The culture method combining ad hesion separation and digestion control is simply performed in need of little co ndition,so it is one of the optimal methods for cell preparation and purificatio n at present.