木犀草素调控视网膜母细胞瘤侵袭性和干样特性的机制研究

Study on regulation mechanism of luteolin for invasion and stem cell-like characteristics of retinoblastoma

目的:探究木犀草素调控视网膜母细胞瘤侵袭性和干样特性的机制。方法:正常培养视网膜母细胞瘤细胞Y79、SO-RB50,以不同浓度木犀草素处理细胞24 h,采用CCK8法检测细胞活力,选取后续给药剂量,采用Transwell小室实验检测细胞侵袭能力,免疫荧光检测细胞中波形蛋白(Vimentin)表达,显微镜下观察干细胞成球能力,Western blot检测细胞中血管内皮生长因子(VEGF)、干细胞标志物(SOX2、OCT4、CD44)、磷脂酰肌醇3-激酶(PI3K)、磷酸化PI3K(p-PI3K)、丝氨酸-苏氨酸蛋白激酶(AKT)、p-AKT、雷帕霉素靶蛋白(mTOR)及p-mTOR表达,构建裸鼠异位肿瘤模型,观察木犀草素对肿瘤生长的影响。结果:与空白组比较,木犀草素干预后Y79、SO-RB50细胞活力明显降低,裸鼠瘤体的生长受到明显抑制(P<0.05),细胞侵袭能力下降,VEGF、Vimentin表达降低(P<0.05),干细胞克隆成球率和成球直径降低,SOX2、OCT4、CD44蛋白表达降低(P<0.05),且细胞中p-PI3K/PI3K、p-AKT/AKT及p-mTOR/mTOR水平也降低(P<0.05)。结论:木犀草素可抑制视网膜母细胞瘤侵袭性和干样特性,可能与抑制PI3K/AKT/mTOR信号通路有关。

Objective:To explore the mechanism of luteolin on regulating the invasion and stem cell-like characteristics of retinoblastoma.Methods:The retinoblastoma cells Y79 and SO-RB50 were normally cultured and treat with different concentrations of luteolin for 24 h. The cell viability was detected by CCK8 to select the subsequent administration doses. The invasion ability of cells was detected by Transwell chamber assay. The expression of Vimentin in cells was detected by immunofluorescence. The spheroidization ability of stem cells was observed under the microscope. The expressions of vascular endothelial growth factor(VEGF),stem cell markers(SOX2,OCT4,CD44),phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),serine-threonine kinase(AKT),p-AKT,mammalian target of rapamycin(mTOR)and p-mTOR in cells were detected by Western blot. The ectopic tumor models of nude mice were constructed to observe the effects of luteolin on tumor growth.Results:Compared with the blank group,activities of Y79 and SO-RB50 were significantly decreased after luteolin intervention,growth of tumors in nude mice was significantly inhibited(P<0.05),invasion ability of cells was decreased,expressions of VEGF and Vimentin were decreased(P<0.05),clone spheroidization rate and spheroidization diameter of stem cells were decreased,expressions of SOX2,OCT4 and CD44 were decreased(P<0.05),and levels of p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR in cells were also decreased(P<0.05).Conclusion:Luteolin can inhibits invasion and stem cell-like characteristics of retinoblastoma,which may be related to inhibiting PI3K/AKT/mTOR signaling pathway.