Notch信号通路在肺癌干细胞中的表达及其对增殖的影响  

Expression of Notch signaling transduction pathway in lung cancer stem cells

作　　者：刘天舒[1] 毛志福[1] 刘军韬[1] 汪巍[1] 毛张凡[1] 黄杰[1] 耿庆[1] 

机构地区：[1]武汉大学人民医院胸外科,430060

出　　处：《中华实验外科杂志》2015年第1期84-87,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81101627）

摘　　要：目的 观察Notch信号通路在肺癌干细胞中的表达及阻断Notch信号通路对肺癌干细胞增殖的影响.方法 以CD133作为肿瘤干细胞表面标志,采用流式细胞仪高速分选技术从人肺腺癌细胞株A549中分离肺癌干细胞及普通肿瘤细胞,实时荧光定量聚合酶链反应（FQ-PCR）及Western blot检测肺癌干细胞和普通肿瘤细胞内的Notch信号通路的表达水平;细胞计数试剂盒（CCK-8）检测肺癌干细胞和普通肿瘤细胞的体外增殖能力,并绘制生长曲线;使用γ-分泌酶抑制剂（DAPT）阻断Notch信号通路传导,观察肺癌干细胞和普通肿瘤细胞的体外生长差异.结果 流式分选前检测CD133阳性细胞（肺癌干细胞）占所有A549细胞的百分比为（0.40±0.11）％,而流式分选后所得细胞中,CD133阳性细胞占百分比为（95.00±0.63）％,两者差异有统计学意义（P＜0.01）.Notch通路在肺癌干细胞及普通肿瘤细胞中均表达,Notch1及Notch2在肺癌干细胞中的表达显著低于在普通肿瘤细胞内的表达,差异有统计学意义（P ＜0.05）;Hes1仅在普通肿瘤细胞中表达,在肺癌干细胞中未检测到表达.肺癌干细胞与普通肿瘤细胞的增殖能力差异无统计学意义（P＞0.05）,而在DAPT干预48 h后,肺癌干细胞和普通肿瘤细胞的体外增殖均受到了抑制,肺癌干细胞的生长抑制率[（33.7±1.9）％]显著高于普通肿瘤细胞的生长抑制率[（21.5±3.4）％],差异有统计学意义（P＜0.05）.结论 较之普通肿瘤细胞,阻断Notch通路传导对肺癌干细胞的生长抑制效果更强,这可能与DAPT抑制了Notch的过高表达对肺癌细胞的负反馈作用有关.Objective To explore the expression of Notch transduction signaling pathway in lung cancer stem cells and the effect on the cellular growth in vitro after blocking Notch signaling pathway.Methods Lung cancer stem cells and general tumor cells were isolated from human lung adenocarcinoma cell line A549 by the high speed sorting of flow cytometry based on surface marker of CD133.Expression of Notch transduction signaling pathway in lung cancer stem cells and general tumor cells were detected by real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） and Western blotting.The proliferation of lung cancer stem cells and general tumor cells in vitro was detected by cell counting kit-8 （CCK-8） respectively,and the growth curves of lung cancer stem cells and general tumor cells were described and compared.After blocking the Notch transduction signaling pathway by γ-secretase Inhibitor （DAPT）,a γ-secretase inhibitor,cellular growth in lung cancer stem cells and general tumor cells was both observed and analyzed.Results CD133 positive cells （served as lung cancer stem cell） occupied （0.40 ± 0.11） % of all A549 cells before the high speed sorting by flow cytometry,while the percentage of CD133 positive cells reached to （95.00 ± 0.63）% after the high speed sorting of flow cytometry,with significant difference （P 〈 0.01）.Homogeneity of lung cancer stem cells could be achieved based on the high speed sorting of flow cytometry.Expressions of Notch transduction signaling pathway were observed in both cancer stem cells and general tumor cells,however significantly lower expression was found in lung cancer stem cells （compared to general tumor cells of lung adenocarcinoma cells,P 〈 0.05）.The growth curves showed that the proliferate potential of lung cancer stem cells in vitro was similar to the proliferation of lung general tumor cells,with no significant difference （P 〉 0.05）.However,after 48 h of DAPT intervention,variant inhibitions of growth were shown.Compared to ge

关 键 词：NOTCH信号通路 肿瘤干细胞 生长抑制 CD133 

分 类 号：R734.2[医药卫生—肿瘤]