摘要目的 持续动态观察胶质瘤干细胞肿瘤生成和参与血管形成过程,研究不同生长阶段皮下移植瘤中干细胞分布与血管的关系.方法 干细胞培养基和免疫磁珠分选法获得C6细胞来源的CD133+细胞.建立CD133+细胞SD大鼠耳朵模型观察胶质瘤干细胞生成肿瘤及促进肿瘤血管形成情况.免疫组化染色研究不同生长阶段移植瘤中干细胞的分布与血管的关系及缺氧诱导因子-1α(HIF-1α)与这种分布的相关性.Western blot法检测各生长阶段肿瘤中CD133蛋白的表达情况.结果 成功分离和鉴定了C6细胞来源的CD133+细胞,并建立了SD大鼠耳朵模型可以实时动态的观察干细胞参与血管形成和肿瘤生成情况.SD大鼠皮下C6胶质瘤模型显示:在肿瘤形成的初期,CD133+细胞在肿瘤内散在分布,随着肿瘤的生长,CD133+细胞开始趋向于微血管,肿瘤生长的后期CD133+细胞在血管周成簇分布；同时随着肿瘤的生长,CD133蛋白的表达也随之增强,Western blot 检测接种后6、9、12、15、20d肿瘤中CD133表达量的吸光度值分别为0.208±0.004、0.282±0.003、0.360±0.004、0.564±0.135、0.756±0.007,各区域间的差异有统计学意义(F=2601.681,P＜0.01).免疫组化染色显示接种6、9、12、15 d皮下移植瘤中CD133的表达分值为0.8±0.4、2.4±0.5、4.0±0.7、6.0±0.7,HIF-1α的分别为0.8±0.4、2.8±0.8、5.0±0.7、6.8±0.4；两者的表达量呈正相关(r =0.921,P＜0.01).结论 胶质瘤干细胞比非干细胞更易形成肿瘤及参与肿瘤血管的形成,HIF-1α及其下游基因产物可能介导了胶质瘤干细胞围绕血管周分布.

abstractsObjectives To dynamically observe how glioma stem cells promote the tumor formation and angiogenesis,and to study the correlation between the distribution of glioma stem cells and mircovessels within different growth stages of subcutaneous tumor.Methods Stem cell medium culture and magnetic activated cell sorting were carried out to obtain CD133+ cells from C6 rat glioma cell line.Sprague Dawley (SD) rat ears model were established to observe glioma stem cells promoting blood vessel formation.Subcutaneous glioma model of C6 and immunohistochemical staining of hypoxia inducible factor-1α(HIF-1α) and CD133 were used to investigate the relationship between distribution of glioma stem cells and mircovessels.Expressions of CD133 protein in each stage of the subcutaneous tumor were detected by Western blot.Results Isolation and identification of glioma stem cells deprived from C6 glioma cell line successfully,the establishment of ears model showed real-time dynamic observation of CD133+ cells involved in angiogenesis and tumor formation. SD rat model of subcutaneous glioma showed the initial of tumor formation,CD133 + cells scattered.With tumor growth,CD133 + cells began to tend to capillaries,in late distributed clusters in perivascular.Meanwhile as tumor growth,CD133 protein expression was gradually increased:the values of Western blot analysis of CD133 expression on 6,9,12,15,20 d were 0.208 ±0.004,0.282 ± 0.003,0.360 ± 0.004,0.564 ± 0.135,0.756 ± 0.007,the differences were significant between different groups ( F =2601.681,P ＜ 0.01 ).At a high magnification,the CD133 scores with immunohistochemical staining on 6,9,12,15 d were 0.8 ± 0.4,2.4 ± 0.5,4.0 ± 0.7,6.0 ± 0.7 ; HIF-1α scores were 0.8 ± 0.4,2.8 ± 0.8,5.0 ± 0.7,6.8 ± 0.4.By Spearman rank correlation analysis found that the relationship between CD133 and HIF-1α expression was positively correlated ( r =0.921,P ＜ 0.01 ).Conclusions Glioma stem cells promote angiogenesis more than non-stem cells; HIF-1α and its downstream gene product might mediate the distribution of glioma stem cells around the perivascular.