低氧条件下视网膜母细胞瘤细胞表达肿瘤干细胞标志物及向内皮样细胞诱导分化的体外研究  

In vitro study on expression of tumor stem cell biomarker and transdifferentiation towardsendothelial cells of retinoblastoma cells under hypoxia condition

作　　者：张丽娜[1] 赵桂秋[1] 王谦[1] 牛膺筠[1] 徐强[1] 杨文毅[1] 

机构地区：[1]青岛大学医学院附属医院眼科,266003

出　　处：《中华眼科杂志》2013年第8期736-743,共8页Chinese Journal of Ophthalmology

摘　　要：目的探讨低氧微环境下人视网膜母细胞瘤（RB）细胞表达肿瘤干细胞标志物及向内皮细胞诱导分化的能力与相关机制。方法实验研究。将人RB细胞株Y79细胞分为正常组、低氧组和预处理组。显微镜下观察3组细胞的形态变化，流式细胞仪检测RB细胞标志物神经元特异性烯醇化酶（NES）及肿瘤干细胞标志物ATP结合盒膜转运蛋白G2（ABCG2）的表达情况，免疫印迹法和实时荧光（real—time）PCR法检测低氧诱导因子-1α（HIF-lα）的表达情况。向内皮细胞诱导分化后应用免疫荧光法检测CD31、vWF的蛋白表达情况，吞噬实验检测其吞噬ae—LDL的能力，三维培养基中观测其血管生成拟态（VM）形成能力，免疫印迹法检测HIF—lα、生促红素人肝细胞A2（EphA2）、磷酸肌醇化激酶3（P13K）的蛋白表达情况。各组问总体差异的比较采用单因素方差分析，进一步的两两比较采用SNK-q检验。结果显微镜下观察结果显示，正常组Y79细胞呈单个或团聚状悬浮生长，低氧组部分细胞伸出突起贴壁生长，预处理组未见贴壁生长的细胞。各组细胞NES及ABCG2染色阳性率差异有统计学意义（F/NES=698．45，FA/ABCG2=864．48，P值均〈0．01）；与正常组[（98．2±2．5）％，（2．1±2．1）％]比较，低氧组NES阳性率[（35．1±3．4）％]明显降低（q=46．11，P〈0．01），ABCG2阳性率[（67．4±3．6）％]明显升高（q=50．89，P〈0．01）；预处理组与正常组NES和ABCG2蛋白表达的差异均无统计学意义（P〉0．05）。与正常组（蛋白：0．165±0．056，mRNA：0．927±0．715）比较，低氧组HIF-1α蛋白（1．094±0．077）及mRNA（6．408±0．686）表达量均明显增加（q蛋白=31．810，P〈0．01；q、mRNA：18．40，P〈0．01），预处理组HIF-1α仪蛋白（0．218±0．061）表达无明显变化（q=1．81，P〉0．05），mRNA（7．219±0．591）的表达增加（q=21．12Objective To investigate the capability of RB cells that represent characteristics of tumor stem cell and trans-differentiate towards endothelial cells under hypoxia microenvironment, as well as their mechanism. Methods Experimental research. RB cell line Y79 was cultured in hypoxia environmentwith or without rapamycin treatment. Morphological changes, expression of neuron specipic enolase （NES） and ATP binding cassette transporters G2 （ABCG2）, and expression of HIF-lα protein and mRNA were detected. After been induced toward endothelial cells, expression of CD31 and vWF, up-taking of Dil-acLDL, vasculogenic mimicry （VM） formation in 3D culture and expression of HIF-lα, EphA2 and PI3K were detected. The ANOVA test was performed to compare the differences among groups, and SNK-q test was performed to further comparison. Results Y79 cells in normal oxygen group were single or agminated suspending cells. In hypoxia group,part of Y79 ceils became adherent. No adherent ceils were observed in rapamycin fore-treated group. Positive rates of NES and ABCG2 had significant difference among these three groups （ FNES = 698.45, FABCG2 = 864. 48, all P 〈 0. 01 ）. Compared with normal group [ （98.2 ± 2. 5 ） % , （2. 1 ± 2. 1 ）% , NES positive rate [ （35. 1 ± 3. 4 ）% ] was significantly reduced and ABCG2 positive rate [ （67.4 ± 3.6） % ] was significantly enhanced in hypoxia group （ q = 46. 11, 50. 89 ; both P 〈 0. 01 ）, no significant changes were observed in rapamycin fore-treated group （ P 〉 0. 05 ） . Expressions of HIF-lα protein and mRNA showed significant difference among these three groups （ Fprotei. = 314. 85, FmRNA = 132. O1, all P 〈 0. 01 ）. Compared with normal oxygen group （0. 165 _± 0. 056,0. 927 ± 0. 715 ）, H IF-1 α protein（ 1. 094 ± 0. 077 ） and mRNA （ 6. 408 ± 0. 686 ） were significantly increased in hypoxia group （ q = 31.81,18.40; both P 〈 0. 01 ）, HIF-1 α mRNA（ 7. 219 ± 0. 591 ）was significantly increased but no increased pro

关 键 词：视网膜母细胞瘤 肿瘤干细胞 缺氧诱导因子1 Α亚基 肿瘤标记 生物学 新生血管化 病理性 

分 类 号：R739.7[医药卫生—肿瘤]