摘要
目的观察Notch1信号途径在食管鳞癌EC9706细胞中的激活状态及其对细胞周期的影响。方法通过免疫细胞化学检测Notch1基因在食管鳞癌细胞株EC9706细胞中的表达,并通过免疫荧光方法研究Notch1基因在EC9706细胞中的激活状态。并且利用CCK-8试剂检测食管癌细胞的增殖状态。此外,采用RT-PCR和Western blot技术检测与细胞周期相关基因的表达,最后通过流式细胞仪进一步探索激活的Notch1信号途径对细胞周期的影响。结果转染pcNICD后的食管鳞癌细胞株中发现Notch1基因的表达。免疫荧光结果显示,转染pcNICD后的食管鳞癌细胞株中Notch1信号途径处于激活状态。与未处理和转染pcDNA3.1的EC9706细胞相比,稳定表达NICD的EC9706细胞的生长速率明显受到抑制(P<0.01)。此外,与未处理的和转染pcD-NA3.1的EC9706细胞相比,稳定表达NICD的EC9706细胞的CDK2,cyclin D1和E基因的mRNA和蛋白的表达明显下调(P<0.05)。转染pcNICD的EC9706细胞在G0/G1期的比率高达74.5%,而未处理的和转染pcDNA3.1的EC9706细胞在G0/G1期的比率分别为59.1%和59.0%。细胞周期分析显示瞬时表达NICD的EC9706细胞在G0/G1期比率的增加,提示激活的Notch1信号途径能够诱导细胞静止在G0/G1期。结论Notch1信号途径的激活引起食管鳞癌细胞的细胞周期静止,提示Notch1基因有可能成为治疗食管鳞癌的新靶点。
Objective To investigate the active status of Notch1 signaling pathway in esophageal squamous cell carcinoma （ESCC） and its effect on cell cycle. Methods The expression of Notch1 gene was detected by
immunocytochemistry in EC9706 cells, and the active status of Notch1 signaling pathway was investigated by immunoflurescence. In addition, cell proliferation assay was performed using CCK-8 Kit. Proteins and mRNA of the genes related to cell cycle was studied by Western blot
and RT-PCR techniques. Finally, cell cycle distribution was measured by flow cytometry （FCM）. Results The expression of Notch1 gene was found in EC9706 cells transfected with pcNICD. Besides, the result of
immunoflurecence revealed that Notch1 appeared to be expressed in the cytosol and nucleus, implying an activated status. EC9706 cells had an obvious decreased growth rate compared to the control EC9706 cells and
EC9706 cells transfected with pcDNA3.1 （P〈0.01）. Furthermore, the mRNA and proteins expressions of the CDK2, cyclin D1 and cyclin E genes were significantly decreased in the EC9706 cells transfected with pcNICD
compared to those of the cells untreated and transfected with pcDNA3.1. There were higher proportions of cells （74.5%,P〈0.05）in EC9706 cells transfected with pcNICD at G0/G1 phase than that in cells untreated （59.1%） and transfected with pcDNA3.1 （59.0%）. Cell cycle distribution analysis demonstrated that the cell increased （P〈0.05）at G0/G1 phase in the cells transiently expressing NICD, which suggested that active Notch1 signaling pathway induces G0/G1 cell cycle arrest in EC9706 cells.Conclusion Active Notch1 signaling pathway can obviously inhibit the growth of EC9706 cells, suggesting Notch1 gene may be a new target
of treating esophageal squamous cell carcinoma.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2009年第11期908-913,共6页
Cancer Research on Prevention and Treatment
基金
河南省医学科技攻关资助项目(20050009)
