摘要
背景：骨髓间充质干细胞分离培养方法的不同可导致其活性与纯度各异，直接影响组织工程的修复效果。目的：对骨髓间充质干细胞体外分离培养条件进行优化组合，并验证其获取骨髓间充质干细胞的效果。设计、时间及地点：细胞学体外实验，于2007-10／12在山西医科大学第二医院骨科实验室完成。材料：清洁级3月龄新西兰白兔2只，由山西畜牧研究所提供。方法：向含有新鲜骨髓的DMEM培养液中加入等量淋巴细胞分离液，采用密度梯度离心和差速贴壁法分离培养兔骨髓间充质干细胞，每间隔8h将未贴壁细胞转移入新的培养瓶，接种5d后首次换液，细胞融合率达90％时胰酶消化传代，第1、3、5代细胞用于实验。主要观察指标：镜下观察细胞形态及生长状态，绘制细胞生长曲线，测定倍增时间，流式细胞仪检测细胞表面抗原CD44标记率。结果：原代培养的骨髓间充质干细胞呈圆形、梭形、多角形等，48h可见贴壁细胞有伸展现象，呈梭形，多角形，成纤维细胞样展开，细胞核清晰，首次换液后可见细胞透亮并充分展开，14d左右可达90％融合。第1、3、5代骨髓间充质干细胞整体呈S型，1～3d为潜伏期，3d后进入对数生长期，7～8d为平台期，平均倍增时间为24．22h。第2代骨髓间充质干细胞CD44呈阳性表达，标记率为93．0％。结论：采用密度梯度离心联合差速贴擘、离心介质选择淋巴细胞分离液、接种5d首次换液的体外分离培养纯化方法，获得的骨髓间充质干细胞生长状态良好，且纯度较高。
BACKGROUND： Different isolation and culture methods for bone marrow mesenchymal stem cell （BMSCs） will result in varying cell activity and purity, which will influence repair effect of tissue engineering. OBJECTIVE： To investigate the optimized methods of isolation and culture of BMSCs in vitro and validate the efficacy Of cell acquisition. DESIGN, TIME AND SETTING： In vitro cytology trial was performed at the laboratory of Department of Orthopedics, Second Hospital of Shanxi Medical University from October to December 2007. MATERIALS： Two 3-month-old New Zealand rabbits were provided by Shanxi Institute of Livestock. METHODS： Lymphocyte isolation solution was added to DMEM culture solution containing flesh bone marrow. BMSCs were obtained and purified by gradient centrifuge and adhesion culture in vitro. Non-attached cells were moved to new culture flask every 8 hours. The solution was changed firstly after 5 days of culture. Trypsinization was conducted at cell confluence of 90%. The first, third and fifth passages of cells were harvested. MAIN OUTCOME MEASURES： The morphology of BMSCs was observed with phase contrast microscope; the growth curve was drawn to determine doubling time. The percentage of the well growth P2 cells were identified by CD44 staining by flow cytometry. RESULTS： Primary cultured BMSCs were oval, spindle-shaped or polygonal, and adhered to plastic surface within 48 hours, exhibiting spindle-shaped or polygonal with clear nucleus, and reached 90% confluence within 14 days. The first, third and fifth passages of BMSCs showed S-type, and were in latent phase at 1-3 days, in log phase 3 days later, and cells came into platform phase at 7-8 days. The mean doubling time was 24.22 hours. CD44-positive cells were found in the second passage, with positive rate of 93.0%. CONCLUSION： BMSCs are easily isolated and cultured in vitro with good growth and high purity by gradient centrifuge and adhesion culture with lymphocyte isolation solution.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第43期8463-8467,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
山西省国际技术合作项目(2007081037)~~
