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

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=21．12，P〈0．05）。向内皮细胞诱导分化实验结果示，低氧组Y79细胞表达CD31、vWF，并获得ac—LDL吞噬能力及VM形成能力，HIF-1α、EphA2及P13K蛋白（1．440±0．089、0．377±0．056、0．762±0．090）表达水平较正常组（0．327±0．108、0．194±0．033、0．402±0．068）明显增加（q=8．72—23．00，P值均〈0．01）。结论低氧条件下部分RB细胞表达肿瘤干细胞标志物，能够向内皮细胞诱导分化从而获得部分内皮细胞表型及功能，并能在三维培养基上形成管道样VM结构；HIF-1α通过EphA2／P13K信号通路在其中发挥重要作用。

Objective 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 protein expression （0. 218 ± 0. 061 ） was observed in rapamycin fore-treated group. After transdifferentiating induction, Y79 cell in hypoxia group expressed CD31 and vWF, acquired the abilities of acLDL up-taking and VM formation. Compared with normal group （0. 327 ± 0. 108, 0. 194 ± 0. 033, 0. 402 ± 0. 068 ） , expression of HIF-lα, EphA2 and PI3K protein （ 1. 440 ± 0. 089,0. 377 ± 0. 056,0. 762 ± 0. 090 ） was significant increased in hypoxia group （ q = 8.72 - 23.00, all P 〈 0. 01 ）. Conclusions Under hypoxie condition, part of RB cells can express tumor stem cell markers, transdifferentiate towards endothelial cells and acquire part of endothelial cell function and VM formation capability. HIF-1 a through EphA2/PI3K pathway may play an important role in VM formation.