摘要
目的:探讨喉癌Hep-2细胞系中CD133+肿瘤干细胞的自我更新机制。方法:流式细胞仪分选Hep-2细胞系中CD133+肿瘤细胞,MTT法测定CD133+肿瘤细胞的自我更新能力;Western blot及RT-PCR法测定自我更新相关基因mRNA及蛋白的表达情况。结果:分选前CD133+细胞比例为(3.10±0.21)%,流式细胞仪分选后纯度可达(90.20±5.51)%,体外培养及生长曲线显示分选后的CD133+细胞增殖速度明显较CD133-细胞快,二者比较差异有统计学意义(P<0.01);CD133+细胞中抗凋亡基因Fas、c-myc、survivin mRNA及蛋白表达量均高于CD133-细胞;同时Bcl-2/Bax比例在CD133+细胞中显著升高;CD133+细胞中β-catenin、SHH、SMOH及Bmi-1、Gli-1无论在mRNA水平还是蛋白水平均表达上调,PTCH表达下调。结论:CD133+Hep细胞具有强的增殖能力,抗凋亡基因表达上调是其自我更新的物质基础;干细胞相关信号通路Wnt、Hedgehog以及Bmi-l信号通路在CD133+中处于活化状态,靶向这些信号通路,有望有效杀伤喉癌干细胞。
Objective：To investigate the self-renewal mechanism of CD133^＋cancer stem cells from Hep-2cell line.Method：The CD133^＋cells were sorted by flow cytometry from Hep-2cell line.Then the sorted CD133^＋cells were cultured in RPMI1640.The ability of self-renewal of CD133^＋cells were tested by MTT assay.mRNA and protein expression of self-renewal related genes were detected by western blot and RT-PCR.Result：（3.10±0.21）%of Hep-2cells expressed the membrane antigen CD133.CD133^＋fraction was raised to（90.20±5.51）%by flow cytometry.In vitro culture and growth curve showed CD133^＋cells had more active proliferation ability than CD133-cells,which showed statistically significant difference between these two group（P〈0.01）.RT-PCR and western blot results showed upregulated mRNA and protein expression of Fas,c-myc,survivin in CD133^＋group（P〈0.01）.In the same time,the ratio of Bcl-2/Bax gene expression was obviously increased in CD133^＋group.Self-renewal related gene such asβ-catenin,SHH,SMOH and Bmi-1,Gli-1were all up-regulated in CD133^＋group both in mRNA and protein.On the contrary,PTCH gene was down-regulated.Conclusion：CD133positive cells are a small proportion of a Hep-2cell line.The results of this experiment verified that CD133 positive cells owned the properties of cancer stem cells.Upregulated anti-apoptotic gene is the foundatiom of self-renewal mechanism of CD133^＋cells.Cancer stem cells related signal pathways such as Hedgehog,Wnt and Bmi-l pathway are in state of activation.The identification of self-renewal mechanism about cancer stem cell provides a powerful tool to investigate the tumorigenic process in the larynx and to develop therapies targeting to these signal pathways.
出处
《临床耳鼻咽喉头颈外科杂志》
CAS
北大核心
2014年第21期1636-1641,共6页
Journal of Clinical Otorhinolaryngology Head And Neck Surgery
基金
国家自然科学基金资助项目(No:81060224)
甘肃省自然科学基金资助项目(No:1010RJZA167)
兰州市科技局立项课题(No:2013-3-31)
关键词
肿瘤干细胞
自我更新
抗凋亡基因
信号通路
cancer stem cells
self-renewal
anti-apoptotic gene
signal pathway
