摘要
目的对兔骨髓间质干细胞进行体外分离培养并观察其生物学特性。方法无菌条件下抽取健康幼年新西兰兔胫骨骨髓2ml,经密度梯度离心后获取骨髓间质干细胞,接种至100ml玻璃培养瓶中进行体外培养,观察其细胞形态、贴壁率和超微结构,流式细胞仪检测细胞表面标记物及细胞生长周期,并进行成骨诱导反向证明,对细胞体外的生物学行为进行研究。结果兔骨髓间质干细胞在体外培养2h后开始贴壁,20h后细胞基本贴壁完全,15d左右细胞融合达90%以上。透射电镜观察显示分离培养的兔骨髓间质干细胞表面可见许多微绒毛,主要分布于胞体一侧,胞核不规则,有切迹,细胞器丰富,具有典型未分化细胞特点;流式细胞仪检测显示分离培养的兔骨髓间质干细胞表面标记物CD44、CD90表达呈阳性,CD45表达呈阴性,且绝大多数(约占细胞总数的97%)细胞的生长周期处于G1或G2期;成骨性诱导3周后,骨髓间质干细胞逐渐形成小的钙盐结晶体,细胞碱性磷酸酶染色阳性,VonKossa染色显示细胞聚集处存在大量钙盐,呈黑色沉积。结论密度梯度离心法获取的兔骨髓间质干细胞纯度较高,生长稳定,传代多次后仍保持干细胞特性,因取材及体外扩增较简单易行,适合作为组织工程椎间盘髓核退变缺损修复的种子细胞来源。
Objective To isolate and culture the rabbit mesenchymal stem cells, and explore the biological features in vitro. Methods Two milliliter of hone marrow was extracted from the tibia of juvenile rabbit under anesthesia and sterile conditions, and mesenchymal stem cells were obtained by density gradient centrifugation and inoculated to 100ml culture flask. The morphological changes, adherence rate and uhrastructure of the stem cells were observed. The surface markers and growth cycle of the mesenchyrnal stem cells were detected by flow cytometry, and biological features were observed by inducing the cells differentiate into osteoblasts. Results The adherence of mesenchymal stem cells started 2 hours after culturing, basically finished 20 hours later, and more than 90% of the cells were fused at the 15th day, Observed with transmission electron microscopy, the cultured cells showed typical ultrastructure of stem cells： numbers of mi crovillus mainly distributed on one side of the cells, irregular nuclei with indentation, and lots of cell organs. Analysis for the cell surface markers indicated positive expression of CD44 and CD90 and negative expression of CD45 in mesenchymal stem cells. Cell growth cycle as say showed that most mesenchymal stem cells （about 97%） stayed in stage G1 and G2. Three weeks after osteoblastic induction, the mes enchymal stem ceils gradually produced small calcium salt crystals, the cells were positive in alkaline phosphatase staining, and Von Kossa staining showed many calcium salts in the ceil clone as hlack reaction deposits. Conclusions The rabbit hone marrow mesenehymal stem cells isolated by density gradient centrifugation are of high purity and stable growth rate, and the cells＇ characters can be still maintained af ter serial subcultivation. Since easy to operate on isolation and proliferation, the mesenchymal stem cells could be used as the tissue engi neering seed cells in repairing of intervertebral disc degeneration.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2008年第10期1235-1238,共4页
Medical Journal of Chinese People's Liberation Army
基金
重庆市自然科学基金资助项目(CSTC
2007BB5056)
关键词
间质干细胞
生物学特性
超微结构
细胞分离
细胞
培养的
mesenchyrnal stem cells
biological features
ultramicrostructure
cell separation
cells, cultured
