摘要
目的建立一种体外分离纯化和培养扩增人骨髓间充质干细胞(mesenchymalstemcells熏MSCs)的方法,并探讨其生物学特性,为MSCs的进一步应用研究提供数据。方法抽取人骨髓后用Percoll分离液经密度梯度离心法分离得到单个核细胞熏以105个细胞蛐cm2密度接种,培养液为含10%胎牛血清的L-DMEM。倒置相差显微镜及吉姆萨-瑞氏染色观察其形态,MTT法测其生长曲线、流式细胞仪检测细胞周期及表面标记物。结果培养的MSCs12h后换液,有部分细胞贴壁生长熏4～5d可见有集落形成熏14～20d左右可达到80%～90%融合熏倒置相差显微镜及吉姆萨-瑞氏染色可见细胞形态为梭形、多角形及纺锤状等熏生长曲线图显示MSCs增殖活跃,流式细胞仪检测显示约有86%的细胞处于G0、G1期,表面标记物中CD14、CD34、VLA-1、CD45和HLA-DR阴性,而CD44、CD105和CD29阳性。结论成功地建立了一种分离培养人骨髓MSCs的方法熏获得的细胞形态多样、增殖稳定。
Objective To establish a method for the isolation and culture of mesenchymal stem cells (MSCs) from human bone marrow in vitro and explore their biological properties. Methods Mononuclear cells were obtained from 3～5 ml of human bone marrow by density gradient centrifugation with Percoll solution and were inoculated into L-DMEM medium containing 10% fetal bovine serum. The configuration was observed under phase-contrast microscope and after staining by Giemsa-Wright method. The cell growth was measured by MTT method, cell cycle and surface antigens were detected through flow cytometer. Results MSCs began to be adhesive after culture for 24h. Formation of colony could be observed on day 4～5. More than 80% MSCs were fused on about day 20, which had diverse configurations under phase-contrast microscope and after Giemsa-Wright staining . About 86% of MSCs were found at G0, G1 phase by flow cytometry. Among surface antigens, CD14,CD34,VLA-1,CD45 and HLA-DR were negative, while CD44,CD105 and CD29 were positive. Conclusion The method for isolation and culture of MSCs has been successfully established in this study and MSCs are of multiform, steady proliferation.
出处
《浙江医学》
CAS
2004年第5期328-330,共3页
Zhejiang Medical Journal
