粉防己碱对神经母细胞瘤干细胞增殖和干性基因表达的影响及作用机制  

Inhibitory effects and mechanisms of tetrandrine on proliferation and stemness marker expression of neuroblastoma stem cells

作　　者：谢东可 何霞 廖凯男 Xie Dongke;He Xia;Liao Kainan(Department of Pediatric Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China)

机构地区：[1]西南医科大学附属医院小儿外科,四川省泸州市646000

出　　处：《中国组织工程研究》2019年第5期729-733,共5页Chinese Journal of Tissue Engineering Research

基　　金：西南医科大学校级课题(2015-YJ092);项目负责人:谢东可~~

摘　　要：背景:研究发现粉防己碱能通过下调β-catenin表达抑制神经胶质瘤干细胞特性,然而其对神经母细胞瘤干细胞的影响未见报道。目的:探讨粉防己碱对神经母细胞瘤干细胞增殖及干性标志物表达的影响,并探究其分子机制。方法:使用无血清悬浮法从神经母细胞瘤SK-N-SH细胞中获取肿瘤干细胞,给予粉防己碱干预,通过MTT法检测粉防己碱对神经母细胞瘤干细胞的增殖抑制作用,细胞免疫荧光染色检测粉防己碱干预后Nestin蛋白水平变化,克隆球形成实验检测粉防己碱干预后肿瘤干细胞成球能力变化,qRT-PCR法检测粉防己碱干预后CD133、Oct-4、Nestin mRNA表达,Western blot检测粉防己碱干预后CD133、Oct-4、Nestin、p-GSK3β和β-catenin蛋白表达。结果与结论:与未干预组比较,粉防己碱干预后SK-N-SH干细胞增殖能力降低(P <0.05),CD133、Oct-4和Nestin mRNA表达降低(P <0.05),Nestin蛋白荧光强度减弱,克隆球形成能力减弱(P <0.05),CD133、Oct-4、Nestin、p-GSK3β和β-catenin蛋白减少(P <0.05)。结果表明,粉防己碱抑制神经母细胞瘤干细胞增殖并降低干性标志物表达,可能与其抑制Wnt/β-catenin信号通路有关。BACKGROUND:Studies have shown that tetrandrine can inhibit the characteristics of glioma stem cells by down-regulating the expression ofβ-catenin.However,its effect on neuroblastoma stem cells has not been reported.OBJECTIVE:To explore the function and mechanism of tetrandrine on proliferation and stemness marker expression of neuroblastoma stem cells.METHODS:Serum-free suspension method was used to obtain SK-N-SH stem cells.SK-N-SH stem cells were treated with tetrandrine,and the inhibitory effect of tetrandrine on the proliferation of neuroblastoma stem cells was detected by MTT method.Immunofluorescence staining was used to detect Nestin expression in the cells after intervention with tetrandrine.The tumorsphere-forming ability of SK-N-SH stem cells was determined by tumorsphere-forming assay.The mRNA levels of CD133,Oct-4and Nestin were tested by qRT-PCR.The protein level of CD133,Oct-4,Nestin,p-GSK3βandβ-catenin were tested by western blot.RESULTS AND CONCLUSION:Tetrandrine inhibited the proliferation of SK-N-SH stem cells(P<0.05).After treated with tetrandrine,the mRNA levels of CD133,Oct-4and Nestin in SK-N-SH stem cells were significantly decreased(P<0.05).Fluorescence intensity of Nestin protien was weakened and the tumorsphere-forming ability of SK-N-SH stem cells was abated(P<0.05).Tetrandrine also inhibited the protein levels of CD133,Oct-4,Nestin,p-GSK3βandβ-catenin(P<0.05).In conclusion,tetrandrine inhibits the proliferation and stemness marker expression of neuroblastoma stem cells,and the mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.

关 键 词：神经母细胞瘤 肿瘤干细胞 粉防己碱 细胞增殖 CD133 OCT-4 NESTIN Wnt/β-catenin信号通路 苄异喹啉类 组织工程 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]