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目的:应用离体培养法培养骨髓来源的血管内皮祖细胞(endothelialprogenitorcells,EPCs)并动态观察扩增效能。方法:2004-04/12在首都医科大学宣武医院外科实验室完成。使用条件培养基和纤维连接蛋白培养骨髓单个核细胞,相差显微镜观察形态变化,测量生长曲线观察增殖能力,检测摄取Dil-ac-LDL的功能,不同时间点行flk-1,CD133和Ⅷ因子的免疫细胞化学检测和CD34,VEG-FR-2及CD133的流式细胞仪检测以定性并观察扩增效率。结果:集落样生长的贴壁细胞平均10d汇合,每毫升骨髓可获得大约(1.3±0.3)×106个细胞,外观鹅卵石样,能摄取Dil-ac-LDL,flk-1,CD133和Ⅷ因子,免疫细胞化学染色均呈阳性,流式细胞仪示VEGFR-2和CD133双阳性细胞扩增达33倍。结论:离体扩增培养法可成功地从骨髓中扩增EPCs,扩增效率能够满足组织工程血管对种子细胞的需要,也为骨髓单个核细胞移植治疗组织缺血提供了间接证据。
OBJECTIVE: To culture bone marrow-derived endothelial progenitor cells (EPCs) by using in vitro culture and observe the amplification efficiency dynamically. Methods: 2004-04 / 12 was completed at Xuanwu Hospital Surgical Laboratory, Capital Medical University. Conditioned medium and fibronectin were used to culture bone marrow mononuclear cells. Morphological changes were observed by phase contrast microscopy. The growth curve was measured to observe the proliferative ability. The function of uptake of Dil-ac-LDL was detected. The expressions of flk-1, CD133 and factor Ⅷ at different time points Immunocytochemistry and flow cytometry analysis of CD34, VEG-FR-2 and CD133 were performed to characterize and observe the amplification efficiency. Results: The adherent cells grew on the average of 10d after colony formation. About 1.3 ± 0.3 × 106 cells were obtained per ml of bone marrow. The appearance of cobblestones was similar to Dil-ac-LDL, flk-1, CD133 and factor Ⅷ, Immunocytochemical staining was positive, flow cytometry showed double positive VEGFR-2 and CD133 cells up to 33 times. CONCLUSION: EPCs can be successfully amplified from bone marrow by ex vivo expansion method. The efficiency of amplification can meet the needs of tissue-engineered blood vessels for seed cells and provide indirect evidence for the treatment of tissue ischemia by bone marrow mononuclear cell transplantation.