摘要
目的研究全反式维甲酸（ATRA）对胶质瘤干细胞（GSCs）N管内皮细胞生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）表达的影响。方法从人胶质母细胞瘤细胞系U87中分离培养GSCs，免疫荧光染色检测CD133、巢蛋白（nestin）的表达进行鉴定。将取GSCs分成3组分别培养：（1）ATRA组：培养基中含10nmol／LATRA；（2）空载体组：培养基中含与ATRA组等量的二甲基亚砜（DMS01；（3）对照组：单纯培养基，培养10d后免疫荧光染色检测胶质纤维酸性蛋白（GFAP）、B．微管蛋白Ⅲ（B．tubulinⅢ）、半乳糖脑苷脂（GalC）的表达；CCK8法检测各组细胞的增殖；EuSA和RT．PCR分别检测各组GSCsVEGF、bFGF的分泌水平和mRNA的表达。结果二代细胞球表达神经干细胞（NSCs）的标记抗体CD133和nestin。免疫荧光染色检测显示分化后GSCs能够分化为多种同源子代细胞（分别表达星形胶质细胞、神经元、少突胶质细胞标志物GFAP、β-tubulinⅢ、Galc）；培养10d后ATRA组细胞GFAP的阳性表达率高于对照组及空载体组，差异有统计学意义（P〈0．05）。第3～7天ATRA组细胞增殖速度较对照组和空载体组明显变缓，差异有统计学意义（P〈0．05）：分化24h后ATRA组GSCsVEGF、bFGF的分泌水平和mRNA的表达均少于对照组和空载体组，差异有统计学意义（P〈0．05）。结论ATRA能诱导GSCs分化并抑制其增殖，其可能通过抑制VEGF和bFGF的表达发挥抗胶质母细胞瘤的作用。
Objective To investigate the effect ofall-trans retinoic acid （ATRA） on expressions of vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF） in glioma stem cells （GSCs）. Methods GSCs were isolated from human glioblastoma cell line U87 and identified by detecting the expressions of CD133 and nestin with immunofluorescence staining. The obtained GSCs were divided into control group, empty vector group （cultured with dimethyl sulfoxide [DMSO]） and ATRA treatment group （cultured with 10 nmol/L ATRA）. After 10 d of differentiation; the proliferation of the treated GSCs was evaluated using CCK8 assay; the expressions of glial fibrillary acidic protein （GFAP）, 13-mbulin Ⅲ and galactocerebroside （GalC） in the cells were detected by immunofluorescence. VEGF and bFGF levels in cultured supernatant were measured by ELISA; the mRNA expressions of VEGF and bFGF were detected by RT-PCR. Results The target antibodies of neural stem cells （NSCs）, CD133 and nestin, positively expressed in the GSCs; differentiated GSCs can differentiate several kinds of homologous daughter cells, which expressed the cell markers of astrocytes, neurons and oligodendrocytes： GFAP, [^-tubulin III and GalC, respectively. The percentage of GFAP-positive differentiated GSCs in the ATRA treatment group was significantly higher as compared with that in the other 2 groups after 10 d of differentiation （P〈0.05）; the speed of proliferation of GSCs in ATRA treatment group was obviously slower than that in the other 2 groups 3-7 d after differentiation （P〈0.05）. The VEGF and bFGF levels and the mRNA expression levels of VEGF and bFGF in GSCs of the ATRA treatment group 24 h after differentiation were also significantly lower than those in the other 2 groups （P〈0.05）. Conclusion ATRA can induce the differentiation of GSCs and inhibit its proliferation. It may exerts its anti-glioblastoma effect through the VEGF and bFGF signaling pathways.
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2012年第6期559-564,共6页
Chinese Journal of Neuromedicine
基金
国家临床重点专科建设项目
