本文選題：鼻咽癌　切入點：EphA2　出處：《中南大學》2013年碩士論文

【摘要】：目的：EphA2是Eph (Erythropoietin-producing hepatoma cell line, Eph)受體酪氨酸激酶家族的成員,具有介導生長因子促進腫瘤發(fā)生和轉(zhuǎn)移的作用。我們前期的定量蛋白質(zhì)組學研究發(fā)現(xiàn),EphA2表達水平及其EphA2S897位點的磷酸化水平在高轉(zhuǎn)移5-8F鼻咽癌(Nasopharyngeal carcinoma, NPC)細胞中上調(diào)。本課題研究EphA2表達水平及其S897位點的磷酸化水平對鼻咽癌細胞增殖、細胞周期分布和細胞遷移能力的影響,并探討EGFR是否通過激活PI3K-AKT使EphA2S897位點磷酸化,為揭示EphA2在NPC發(fā)生和轉(zhuǎn)移中的作用及其機制奠定基礎(chǔ)。 方法：(1)Western blotting檢測高轉(zhuǎn)移的NPC細胞株5-8F細胞和低轉(zhuǎn)移的NPC細胞株6-10B細胞中EphA2表達水平及其EphA2S897位點的磷酸化水平,以及EGFR表達水平和磷酸化EGFR的水平,實時熒光定量PCR檢測兩株細胞中EphA2配體EphrinA1的mRNA表達水平；(2)Western blotting結(jié)合小分子激酶抑制劑檢測EGFR是否通過激活PI3K-AKT使EphA2S897位點磷酸化；(3)脂質(zhì)體轉(zhuǎn)染法將突變型EphA2(S897A)表達載體和空載體轉(zhuǎn)染5-8F細胞,將野生型EphA2表達載體和空載體分別轉(zhuǎn)染6-10B細胞,建立穩(wěn)定轉(zhuǎn)染細胞系；(4)以突變型EphA2高表達的5-8F細胞系、野生型EphA2高表達的6-10B細胞系及其空白載體轉(zhuǎn)染細胞系為樣本,采用MTT法檢測細胞增殖,采用流式細胞儀檢測細胞周期分布,劃痕實驗觀察細胞的遷移。 結(jié)果：(1)5-8F細胞EphA2的表達水平和EphA2S897位點的磷酸化水平以及EGFR的表達水平和磷酸化EGFR的水平明顯高于6-10B細胞,證實了我們前期的定量蛋白質(zhì)組學結(jié)果；(2)EphA2配體Ephrin A1mRNA在5-8F細胞的表達水平低于6-10B細胞；(3)在5-8F細胞和6-10B細胞中,EGFR通過激活PI3K-AKT使EphA2S897位點磷酸化；(4)建立了突變型EphA2高表達的5。8F細胞系,野生型EphA2高表達的6-10B細胞系及其空白載體轉(zhuǎn)染細胞系；(5)與空載體轉(zhuǎn)染的5-8F細胞和未轉(zhuǎn)染5-8F細胞比較,突變型EphA2高表達的5-8F細胞的增殖能力降低,G0/G1細胞增加,而S期細胞減少,細胞遷移能力降低；與空載體轉(zhuǎn)染的6-10B細胞和未轉(zhuǎn)染6-10B細胞比較,野生型EphA2高表達的6-10B細胞增殖能力增加,S期細胞增加,而G2/M期細胞減少,細胞遷移能力增強。 結(jié)論：1.高轉(zhuǎn)移5-8F NPC細胞的EphA2表達水平及其S897位點的磷酸化水平顯著高于低轉(zhuǎn)移的6-1OB NPC細胞,而Ephrin A1的表達水平顯著低于6-1OB NPC細胞；2. EGFR通過激活PI3K-AKT信號通路使NPC細胞EphA2S897位點磷酸化；3.建立了突變型EphA2高表達的5-8F細胞系和野生型EphA2高表達的6-10B細胞系；4.EphA2高表達及其S897位點的磷酸化促進NPC細胞的生長和遷移。
[Abstract]:Objective: EphA2 is a member of the tyrosine kinase family of Eph Erythropoietin-producing hepatoma cell line. Our previous quantitative proteomics studies showed that the expression of EphA2 and the phosphorylation level of EphA2S897 site were up-regulated in high metastatic 5-8F nasopharyngeal carcinoma (NPCs) cells. The aim of this study was to investigate the effect of EphA2 expression and phosphorylation at S897 on the proliferation of nasopharyngeal carcinoma cells. The effects of cell cycle distribution and cell migration ability, and whether EGFR phosphorylates EphA2S897 sites by activating PI3K-AKT are discussed, which lays a foundation for revealing the role and mechanism of EphA2 in the pathogenesis and metastasis of NPC. Methods the expression of EphA2 and the phosphorylation of EphA2S897 site, EGFR expression and phosphorylated EGFR in 5-8F cells with high metastasis and 6-10B cells with low metastasis were detected by Western blotting. Real-time fluorescence quantitative PCR was used to detect the mRNA expression level of EphA2 ligand EphrinA1 in the two cell lines. Western blotting combined with small molecular kinase inhibitor was used to detect whether EGFR phosphorylated EphA2S897 site by activating PI3K-AKT. The mutant EphA2S897A) expression vector was transfected by liposome. The empty vector was transfected into 5-8F cells. The wild-type EphA2 expression vector and empty vector were transfected into 6-10B cell line respectively. A stable transfection cell line was established. The mutant EphA2 overexpression 5-8F cell line, the wild-type EphA2 overexpression 6-10B cell line and its blank vector transfected cell line were used as samples. Cell proliferation was detected by MTT assay, cell cycle distribution was detected by flow cytometry, cell migration was observed by scratch test. Results the levels of EphA2 expression, EphA2S897 site phosphorylation, EGFR expression and phosphorylated EGFR in the 5-8F cells were significantly higher than those in 6-10B cells. It was confirmed that the expression level of EphA2 ligand Ephrin A1mRNA in 5-8F cells was lower than that in 6-10B cells (5-8F cells and 6-10B cells). In 5-8F cells and 6-10B cells, EphA2S897 sites were phosphorylated in 5-8F cells and 6-10B cells by activating PI3K-AKT. Compared with 5-8F cells transfected with empty vector and 5-8F cells transfected with empty vector, the proliferative ability of 5-8F cells with high expression of mutant EphA2 decreased, while that of G-0 / G1 cells increased, and that of S-phase cells decreased, compared with 5-8F cells transfected with wild type EphA2 and its blank vector. Compared with 6-10B cells transfected with empty vector and non-transfected 6-10B cells, the proliferative ability of wild-type EphA2 overexpression 6-10B cells increased in S phase, while that in G _ 2 / M phase decreased and cell migration increased. Conclusion the level of EphA2 expression and phosphorylation at S897 site in high metastatic 5-8F NPC cells were significantly higher than those in 6-1OB NPC cells with low metastasis. The expression level of Ephrin A1 was significantly lower than that of 6-1OB NPC cells. EGFR phosphorylated EphA2S897 sites in NPC cells by activating PI3K-AKT signaling pathway. 5 8F cell lines with high expression of mutant EphA2 and 6-10B cell lines with high expression of wild type EphA2 were established. Phosphorylation of S897 and S897 promoted the growth and migration of NPC cells.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R739.63

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相關(guān)期刊論文 前1條

1 陳瑜;李嬌陽;李茂玉;肖志強;;不同轉(zhuǎn)移潛能鼻咽癌細胞株的差異膜蛋白質(zhì)組研究[J];國際病理科學與臨床雜志;2012年06期

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