兰州大学机构库 >生命科学学院
靶向抑制NOTCH信号通路诱导胃癌细胞凋亡的网络调控研究
Alternative TitleCross-talking of cellular signalling network in gastric cancer cell targeted inhibition of NOTCH signaling pathway
刘涛
Thesis Advisor李玉民
2014-05-26
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keyword幽门螺杆菌 NOTCH信号通路 胃癌 细胞凋亡
Abstract目的 探讨靶向抑制NOTCH信号通路应用于胃癌治疗的可能性,同时阐释抑制该通路诱导胃癌细胞凋亡的机制。 方法 通过基因芯片筛选H. pylori感染后出现异常的信号通路,利用荧光定量PCR和Western Blot验证上一步筛选出的通路。利用流式细胞术检测抑制Notch信号通路对胃癌细胞周期及凋亡的影响,通过基因芯片分析胃癌细胞中NOTCH通路与其它通路的cross-talking,通过凋亡抗体芯片阐释阻断NOTCH通路诱导胃癌细胞凋亡的机制。 结果 H. pylori感染后有22个基因表达水平具有差异,这些基因主要与细胞增殖、细胞周期和转录调控相关,主要涉及Cyclins and Cell Cycle Regulation和NOTCH Signaling Pathway这两条通路。 使用GSI 作用于胃癌细胞株后NOTCH通路受到了显著抑制;抑制剂使得胃癌细胞停滞于G1期,抑制了细胞的增殖,并且诱导胃癌细胞发生凋亡。siRNA能显著抑制相应蛋白表达水平,使靶基因HES1表达水平显著下降,显著诱导了胃癌细胞的凋亡。对GSI、siRNA处理后的MKN-45进行基因表达分析发现,三组无显著差异,NOTCH信号通路与WNT信号通路、P53信号通路和缺氧信号通路密切相关。利用凋亡抗体芯片分别对GSI、siRNA处理后的MKN-45进行蛋白差异表达分析发现,GSI通过死亡受体途径和线粒体凋亡途径诱导胃癌细胞的凋亡;而siRNA仅通过死亡受体途径调控胃癌细胞的凋亡。 结论 NOTCH信号通路可做为胃癌治疗的潜在靶点。
Other AbstractAims The aim of this study was to investigate the possibility of NOTCH signaling pathway as a potential therapeutic target in gastric cancer, and explain the molecular mechanism which inhibition of Notch pathway induces apoptosis in gastric cancer cells. Methods Gastric epithelial cell line GES-1 was co-cultured with H. pylori for 24h. The signaling pathways activated by H. pylori were identified by RT ² Profiler PCR Array - Human Signal Transduction Pathway Finder microarray. Total RNA and protein were isolated from GES-1 incubation with different strains of H. pylori and were cocultured for 24 h. The activated signaling pathway verified by qPCR and Western Blot. Gastric cancer lines (MKN-45, SGC-7901, BGC-823) will be treated with or without GSI, Notch receptor siRNA and ligands siRNA. Cell cycle and apoptosis were detected by Annexin-V-FITC/PI and flow cytometry. Cross-talking between NOTCH signaling pathway and other signaling pathways analyzed by RT ² Profiler PCR Array - Human Signal Transduction Pathway Finder microarray. The molecular mechanism which inhibition of NOTCH pathway induces apoptosis in gastric cancer cells analyzed by Human Apoptosis Antibody Array. Results There are 22 different expression genes between GSI with and without H. pylori 11637 by RT ² Profiler PCR Array - Human Signal Transduction Pathway Finder microarray. These genes were associated with cell proliferation, cell cycle and transcriptional regulation. DAVID Pathway analysis showed that the H. pylori infection mainly affects Cyclins and Cell Cycle Regulation and NOTCH Signaling Pathway. The expression levels of NOTCH ligand DLL4 were significantly increased by H. pylori 11637 and SS1. H. pylori inhibited cell proliferation through the regulation of cell cycle-related proteins. Gastric cancer cell lines (BGC-823, MKN-45, SGC-7901) were treated with or without 20 μmol / L GSI for 24 h. The expression levels of mRNA and protein of NOTCH1, NOTCH2 and HES1 were inhibited significantly. Inhibitor induced cell-cycle arrest in G1 phase, inhibited cell proliferation and induced apoptosis. NOTCH2 siRNA and DLL4 siRNA significantly inhibited the expression of the corresponding protein levels and decreased significantly HES1. siRNA group did not affect gastric cancer cell cycle. Both GSI group and siRNA group induced significantly gastric cancer cells apoptosis. NOTCH signaling pathway closely related to WNT signaling pathway, P53 signaling pathway and...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221793
Collection生命科学学院
Recommended Citation
GB/T 7714
刘涛. 靶向抑制NOTCH信号通路诱导胃癌细胞凋亡的网络调控研究[D]. 兰州. 兰州大学,2014.
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