【作者】 杨青山；

【导师】 樊飞跃； 张春明； 穆传杰；

【作者基本信息】 中国协和医科大学 ， 放射医学， 2008， 博士

【摘要】 食管癌是人类常见的恶性肿瘤之一,其发病率和死亡率均很高。Ku80是一种DNA结合蛋白,主要参与非同源末端连接修复DNA双链断裂,此外,在维持端粒结构的稳定性,免疫球蛋白V（D）J链重排及调控特定基因转录等方面起着重要作用。近年来研究发现,Ku80蛋白与食管癌的发生和发展密切相关。然而关于Ku80蛋白在食管癌发生、发展中的具体机制目前还未见报道。我们通过对26例食管癌标本和10例食管癌旁标本的免疫组织化学分析,发现在食管癌组织中有高水平的Ku80蛋白表达。进一步检测不同肿瘤细胞系及胚肾细胞系中Ku80蛋白表达得到了相似的结果,在不同肿瘤细胞系中都有高水平的Ku80蛋白表达,且以食管癌细胞系EC9706最高。RNAi技术是近年来新发展起来的研究基因功能的强有力工具,本实验成功构建了pGCshRNA-Ku80载体。通过转染食管癌细胞系EC9706,经G418筛选得到了稳定的Ku80基因沉默食管癌细胞系。研究结果表明,Ku80基因沉默对体外食管癌细胞的增殖、粘附和侵袭能力均有抑制作用。体内实验显示,Ku80基因沉默能抑制食管癌细胞裸鼠移植瘤的生长。Ku80基因沉默能提高γ-射线和丝裂霉素C药物的敏感性,促进γ-射线和MMC诱导食管癌细胞发生凋亡,并使细胞周期阻滞于G₂/M期。总之,我们的研究首次为Ku80具有促进肿瘤细胞增殖、粘附和侵袭能力及在食管癌发生、发展起着重要作用提供了实验依据。同时证实了Ku80基因沉默可提高肿瘤细胞对γ-射线和药物的敏感性,为今后应用于临床治疗奠定了理论基础。更多还原

【Abstract】 Esophageal cancer is one of the most common malignant tumors, which morbidity and mortality are high. Ku80 is a DNA-binding protein and mainly joins in non-homologous end joint and repairs DNA double-strand break. Moreover, it plays an important role in maintaining the stability of telomere structure, rearrangement of immunoglobulin V （D） J, controlling specific gene transcription. In recent studies, it is known that Ku80 is closely related to the occurrence and development of esophageal cancer. But the specific mechanism of Ku80 protein in the occurrence and development of esophageal cancer is still not reported.Through immunohistochemical analysis of 26 cases of esophageal cancer specimens and 10 cases of adjacent esophageal cancer specimens, we found that there is a high level expression of Ku80 protein in esophageal cancer tissue. In our experiments, Ku80 protein expressions in different tumor cell lines and embryonic kidney cell line HEK293 were analyzed. There is a high level of Ku80 protein expression in different tumor cell lines, especially in esophageal cancer cell lines EC9706.RNAi technology is a new and powerful tool for researching gene function in recent years. We successfully constructed pGCshRNA-Ku80 vector, and got stable silencing of Ku80 gene of esophageal carcinoma cell line by transfecting esophageal cancer cells EC9706 and selecting with G418.Our research showed that cells proliferation, adhesion and invasion were inhibited in esophageal cancer cells by silencing Ku80 gene in vitro. In vivo experiments, it was showed that silencing Ku80 gene could inhibit tumorigenicity in a xenograft model of nude mice.Silencing Ku80 gene could increase the sensitivity of tumor cells toγ-rays and mitomycin C, which promoted esophageal cancer cell apoptosis induced byγ-rays and MMC. Silencing Ku80 gene made cell arrested in G₂/M phase of cell cycle.In conclusion, it was confirmed firstly that Ku80 could promote esophageal cancer cells proliferation, adhesion and invasion in our experiments. Thus, Ku80 plays an important role in the occurrence and development of esophageal cancer. Moreover, silencing Ku80 gene could increase the sensitivity ofγ-rays and chemicals, which provide a theoretical basis for applying in clinical cancer therapy in the future.更多还原