组蛋白去乙酰化酶抑制剂apicidin对胶质瘤干细胞生物活性的影响  

Effect of histone deacetylase inhibitor apicidin on biological activity of glioma stem cells via down-regulating Nanog expression

作　　者：杨洋 牛朝诗 程传东 李冬雪 李仲颖 汪炎 

机构地区：[1]安徽医科大学附属省立医院神经外科脑功能与脑疾病安徽省重点实验室安徽省脑立体定向神经外科研究所,合肥230001

出　　处：《中华神经医学杂志》2013年第12期1209-1215,共7页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81172407）

摘　　要：目的探讨组蛋白去乙酰化酶抑制剂apicidin介导的Nanog表达抑制对胶质瘤干细胞（GSCs）增殖、迁移和侵袭能力的影响。方法人胶质瘤细胞系U87体外常规培养，应用无血清悬浮培养法获得GSCs，免疫荧光染色检测细胞CD133、巢蛋白（nestin）的表达进行鉴定；以0．5μmol／L apicidin处理GSCs48h作为实验组，以未经处理的GSCs作为空白对照组，RT．PCR和Westemblotting分别检测2组细胞apicidin靶基因NanogmRNA和蛋白的表达；免疫荧光双重染色检测GSCs中CDl33和Nanog蛋白的表达；噻唑蓝（MTT）比色法检测0．2、0．5、1．0、2．0μg／mL apicidin对细胞增殖的抑制作用；Transwell实验检测apicidin对细胞迁移和侵袭能力的影响。结果由U87胶质瘤细胞系成功获得CD133、nestin表达阳性的GSCs：与空白对照组相比，实验组细胞中NanogmRNA和蛋白的表达显著减少，Nanog＋CD133＋以及Nanog＋／CD133＋细胞阳性率均显著降低，细胞的迁移[迁移细胞数：（87．50±4．65）个／视野vs（128．50±6．14）个／视野]和侵袭能力[穿膜细胞数：（55．75±4．79）个／视野口s（81．50_＋5．45）个／视野）1下降，差异有统计学意义（P〈0．051；MTT比色法显示不同浓度apicidin组GSCs细胞的吸光度似）值较空白对照组降低，抑制率增加，而且apicidin浓度越高，GSCs细胞的A值越低，抑制率越高，比较差异均有统计学意义（P〈0．05）。结论组蛋白去乙酰化酶抑制剂apicidin可抑制靶基因NanogmRNA和蛋白水平表达，从而抑制GSCs的细胞增殖、迁移和侵袭能力。Objective To investigate the effect ofhistone deacetylase inhibitor apicidin induced Nanog repression on proliferation, migration and invasion of glioma stem cells （GSCs）. Methods GSCs were isolated from glioma cell line U87 and cultured in simplified serum-free neural stem cell medium by nanosphere suspension culture method, and purified continuously through the monoclonal formation experiment. The immunofluorescence staining of cells was employed to detect the CD133 and Nestin expressions to identify GSCs. Apicidin treatment group （GSCs treated with 0.5 ixmol/L apicidin for 48 h） and blank control group were employed. Nanog mRNA and protein expressions were detected by real time-PCR and Western blotting, respectively. Double immunofluorescence staining was used to detect the co-expressions of Nanog/CD133. MTT assay was used to detect the proliferation modification of GSCs, while migration and invasion of GSCs were detected by Transwell migration and invasion assay after 0.2, 0.5, 1.0 and 2.0 μg/mL apicidin oftreatrnent. Results GSCs were isolated, cultured and purified from glioblastoma cell line U87; as compared with those in the blank control group, the mRNA and protein expressions of Nanog were significantly repressed and Nanog＋, CD133~ and Nanog＋/CD133＋ cells were obviously reduced in the apicidin treatment group （P〈0.05）. The migration and invasion in the apicidin treatment group were inhibited dramatically as compared with those in the blank control group （cell number of migration： [87.50：~4.65]/field vs. [128.50_＋6.14]/field; cell number of transmembrane： [55.75＋4.79J/field vs. [81.50_＋5.45J/field,/9〈0.05）. MTT assay indicated that the absorbance value of the apicidin treatment group was significantly lower as compared with that in the blank control group （P〈 0.05）; the higher the apicidin concentration, the lower the absorbance value and the more obvious the proliferation inhibition. Conclusion Histone deacetylase inhibitor apicidin could inhibit the mRNA and

关 键 词：组蛋白去乙酰化酶抑制剂 NANOG 神经胶质瘤 胶质瘤干细胞 

分 类 号：R739.4[医药卫生—肿瘤]