人骨髓CD34+干细胞分离培养及血管内皮生长因子165诱导分化的作用

背景：有研究证实，骨髓中CD34+干细胞在一定条件下具有向内皮细胞分化的潜能。 目的：体外分离、纯化、培养和扩增人CD34+造血干细胞，并检测其免疫学表型，观察血管内皮生长因子对其体外诱导分化后细胞免疫表型的变化。 设计、时间及地点：细胞学观察实验，于2005-10/2007-10在南方医科大学神经生物学教研室完成。 材料：骨髓来源于健康供者。 方法：利用Percoll 梯度分离、贴壁筛选法及单克隆培养法分离培养、扩增人骨髓CD34+干细胞。采用脂质体介导转染法，选生长良好的传代细胞，以血管内皮生长因子165 基因转染人骨髓CD34+干细胞。 主要观察指标：采用免疫荧光和流式细胞术检测人骨髓CD34+干细胞免疫学表型。经血管内皮生长因子165 基因转染后，人骨髓CD34+干细胞的表型变化以及血管内皮生长因子的分泌情况。 结果：体外分离培养出高度同源性的人骨髓CD34+干细胞，细胞形态呈成纤维细胞样。CD44、CD29和c-kit阳性，CD31和CD54阴性；经血管内皮生长因子165 诱导后，人骨髓CD34+干细胞表面标志CD44 表达降低，CD31升高，呈现典型的内皮细胞表型，并且获得大量分泌血管内皮生长因子的能力。 结论：采用Percoll分离液梯度离心继以贴壁筛选法及单克隆培养法联合筛选分离，可培养扩增出高度同源的CD34+干细胞，经血管内皮生长因子基因转染后，CD34+干细胞呈典型的内皮细胞表型，验证了其具有向内皮细胞分化的潜能。

Culture of human bone marrow CD34+ stem cells and differentiation induced by vascular endothelial growth factor 165

BACKGROUND: CD34+ stem cells in bone marrow can differentiate into endothelial cells under a certain condition. OBJECTIVE: To in vitro separate, purify and culture human marrow CD34+ hematopoietic stem cells, and to detect their immunophenotypes and to investigate the changes in cell immunophenotype after in vitro differentiation by vascular endothelial growth factor (VEGF) transfectant. DESIGN, TIME AND SETTING: The cytology observation experiment was performed at the Institute of Neurobiology, Southern Medical University from October 2005 to October 2007. MATERIALS: The bone marrow was obtained from healthy donors. METHODS: CD34+ stem cells were separated, cultured and proliferated by Percoll gradient centrifugation, adherence screening method and monoclonal culture. Well growth passaged cells were collected by liposome-mediated infection protocol. VEGF165 gene was used to transfect human bone marrow CD34+ stem cells. MAIN OUTCOME MEASURES: Immunophenotypes of CD34+ stem cells were detected by immunofluorescence and flow cytometry. Cell immunophenotype changes and VEGF secretion were detected following VEGF156 gene transfection. RESULTS: High homogenous CD34+ stem cells had been isolated and cultured in vitro, and the morphous was like fibroblast. CD34+ stem cells had unique immunophenotypes and they were positive for CD44, CD29 and c-kit, but negative for CD31 and CD54. After VEGF-165 transfection, CD34+ stem cells presented endothelial differentiation with the expression of CD44 decrease and CD31 increase, as well as could secrete a large number of VEGF. CONCLUSION: High homogenous CD34+ stem cells had been isolated and cultured by Percoll isolation method, adherence screening method and monoclonal culture. VEGF-165 transfectant can induce CD34+ stem cells to differentiate towards endothelial cells.