人脐血源性内皮祖细胞血管发生活性及其参与人恶性胶质瘤细胞异种移植瘤血管新生的作用

目的 观察人脐血源性内皮祖细胞(EPC)血管发生能力和在恶性胶质瘤血管新生过程中的作用.方法 应用密度梯度离心法分离新鲜人脐血的单个核细胞,接种于EGM-2培养液中培养获得EPC.取生长到第7～10天的细胞进行CD34和VEGFR-2免疫荧光双标染色.检测血管内皮生长因子(VEGF)刺激下EPC增殖活性、迁移能力和体外形成小管样结构的能力.采用人恶性胶质瘤细胞系U87在免疫缺陷小鼠进行皮下移植,于接种肿瘤后第7天经尾静脉注射EPC(每只5×103),并于接种肿瘤后第28天取材检测肿瘤微血管和EPC组织分布及定位,采用抗人CD31和抗鼠CD31免疫荧光双标记肿瘤微血管,计算人源性EPC来源的血管占肿瘤血管网的比例.结果 培养的细胞在第7～10天时可见条索样结构,生长并逐渐融合形成铺路石样排列的单层细胞,表达内皮细胞标记物CD34和VEGFR-2.在VEGF刺激下EPC具有较强的增殖活性、迁移能力和体外形成小管样结构的能力.外源性EPC能特异性归巢到异种移植瘤组织并形成新生血管,占肿瘤血管网的(18.68±1.32)%.结论 EPC在体内外具有形成血管能力,并参与异种移植瘤血管新生,提示其在恶性肿瘤血管新生过程中具有重要作用,并可能参与肿瘤微血管构筑表型异质性.

Vasculogenic potential of endothelial progenitor cells derived from human umbilical cord blood and their roles in neovascularization of malignant glioma

OBJECTIVE: To investigate vasculogenic potential of endothelial progenitor cells (EPCs) derived from human umbilical cord blood and their contribution to the neovascularization of malignant glioma in vivo. METHODS: EPCs were isolated from human umbilical cord blood by density gradient centrifugation. After 7-10 days of culture, EPCs were investigated for CD34 and VEGFR-2 expression by direct immunofluoresent staining. The proliferative activity, migratory capability and forming capillary-like tubules were also monitored after stimulation with VEGF(50 mg/L) in vitro. Moreover, EPCs were administered into tumor-bearing mice, and the tumor and mouse organs were examined under confocal laser scanning microscope to visualize the distribution and localization of transplanted EPCs. In order to quantity the incorporation of EPCs into tumor vessels, cryosections of the tumor tissue were double-labelled with antihuman CD31 and anti-mouse CD31. RESULTS: After 7 to 10 days of culture, EPCs assumed cobblestone-like monolayer growth pattern with nearly complete confluence, and expressed CD34 and VEGFR-2. Significant proliferative activity, increased migratory capability and forming capillary-like tubules were observed when stimulated with VEGF. The transplanted EPCs in vivo specifically homed to solid tumor tissue and incorporated into the tumor's endothelium. Quantitative analysis revealed that human EPCs contributed significantly to tumor neovascularization by incorporation into tumor vasculature (18.68 +/- 1.32)% of the total vessels. CONCLUSION: EPCs possess the potential to form neovascular network in tumor and play a role in the phenotypical heterogeneity of tumor microvascular architecture.