本文選題：胰腺癌　切入點(diǎn)：增殖　出處：《華中科技大學(xué)》2016年博士論文

【摘要】：研究目的：在多種惡性腫瘤中,YB-1表達(dá)水平的增高可促進(jìn)腫瘤的發(fā)生發(fā)展。而在胰腺癌中YB-1的表達(dá)情況,是否影響胰腺癌進(jìn)展,特別是其作用機(jī)制,并沒有明確的報(bào)道。本研究旨在探討YB-1在胰腺癌中的表達(dá)及與胰腺癌進(jìn)展的關(guān)系,并深入探討YB-1發(fā)揮生物學(xué)效應(yīng)的分子機(jī)制。實(shí)驗(yàn)方法：本研究通過免疫組織化學(xué)(IHC)檢測(cè)90對(duì)胰腺癌和癌旁組織中YB-1的表達(dá),并分析YB-1的陽(yáng)性表達(dá)與胰腺癌分化程度的相關(guān)性。分析通過western blot法檢測(cè)胰腺癌細(xì)胞系中YB-1的表達(dá)。使用慢病毒沉默技術(shù)抑制胰腺癌細(xì)胞系中YB-1的表達(dá),通過CCK-8法和流式細(xì)胞術(shù)檢測(cè)YB-1沉默對(duì)于胰腺癌細(xì)胞系增殖能力和對(duì)細(xì)胞周期的影響。通過western blot檢測(cè)其對(duì)細(xì)胞周期相關(guān)蛋白表達(dá)的影響。通過western blot法和qPCR法檢測(cè)YB-1沉默的胰腺癌細(xì)胞中GSK-3β蛋白表達(dá)和mRNA表達(dá)的變化。使用慢病毒技術(shù)過表達(dá)胰腺癌細(xì)胞系中的YB-1,通過western blot法檢測(cè)YB-1過表達(dá)的胰腺癌細(xì)胞系中GSK-3β表達(dá)的變化。應(yīng)用GSK-3β抑制劑TWS119,通過CCK-8法和流式細(xì)胞術(shù)檢測(cè)其過表達(dá)以及加用GSK-3β抑制劑對(duì)胰腺癌細(xì)胞增殖能力及細(xì)胞周期的影響,是否能夠逆轉(zhuǎn)過表達(dá)YB-1的作用。應(yīng)用PI3K/AKT、 MEK/ERK信號(hào)通路抑制劑,通過Western blot技術(shù)檢測(cè)在胰腺癌細(xì)胞系中,細(xì)胞信號(hào)通路對(duì)YB-1的調(diào)控的機(jī)制。實(shí)驗(yàn)結(jié)果：YB-1在胰腺癌中顯著高于癌旁組織,且與胰腺癌臨床分化程度呈正相關(guān)。YB-1在胰腺癌細(xì)胞系中高表達(dá)。使用慢病毒沉默胰腺癌細(xì)胞中YB-1的表達(dá)后,可以顯著抑制胰腺癌的增殖,并使胰腺癌細(xì)胞出現(xiàn)G1期阻滯,慢病毒過表達(dá)的胰腺癌細(xì)胞系中,GSK-3β表達(dá)明顯上調(diào),增殖能力顯著增強(qiáng),G1期細(xì)胞比例明顯減少。抑制YB-1后,胰腺癌細(xì)胞中GSK-3β mRNA水平明顯降低,且蛋白表達(dá)水平變化與mRNA水平變化一致；相反,過表達(dá)YB-1后,GSK-3β mRNA與蛋白表達(dá)均表達(dá)明顯上調(diào)。使用GSK-3β特異性抑制劑處理YB-1過表達(dá)的胰腺癌細(xì)胞系后,YB-1過表達(dá)所致的細(xì)胞增殖能力增強(qiáng)被抑制,且CyclinD1、E1蛋白表達(dá)水平與細(xì)胞增殖水平變化一致。應(yīng)用PI3K/AKT及MEK/ERK信號(hào)通路的抑制劑發(fā)現(xiàn),YB-1同時(shí)受這兩條信號(hào)通路調(diào)控,其中PI3K/AKT在轉(zhuǎn)錄水平調(diào)控,MEK/ERK在翻譯水平調(diào)控。研究結(jié)論：YB-1過表達(dá)是胰腺癌具有高度惡性生物學(xué)行為的重要機(jī)制,其通過調(diào)控GSK-3β的表達(dá)促進(jìn)胰腺癌細(xì)胞的增殖與腫瘤進(jìn)展。YB-1和GSK-3β具有成為成為胰腺癌治療的靶點(diǎn)的潛力。
[Abstract]:Objective: to study whether the expression of YB-1 in pancreatic cancer affects the progression of pancreatic cancer, especially its mechanism. The purpose of this study was to investigate the expression of YB-1 in pancreatic cancer and its relationship with the progression of pancreatic cancer. In this study, the expression of YB-1 in pancreatic cancer and adjacent tissues was detected by immunohistochemistry. The expression of YB-1 in pancreatic cancer cell line was detected by western blot method. Lentivirus silencing technique was used to inhibit the expression of YB-1 in pancreatic cancer cell line. The effect of YB-1 silencing on the proliferation and cell cycle of pancreatic cancer cell line was detected by CCK-8 and flow cytometry. The expression of cell cycle related protein was detected by western blot. YB-1 was detected by western blot and qPCR. The changes of GSK-3 尾 protein and mRNA expression in silent pancreatic cancer cells. YB-1 in pancreatic cancer cell line was overexpressed by lentivirus technique, and GSK-3 尾 expression was detected by western blot assay. GSK-3 尾 was used to detect the expression of GSK-3 尾 in pancreatic cancer cell line with YB-1 overexpression. TWS119 was used to detect the overexpression of TWS119 by CCK-8 and flow cytometry, and the effect of GSK-3 尾 inhibitor on the proliferation and cell cycle of pancreatic cancer cells. Whether it can reverse the effect of overexpression of YB-1. Using PI3K / AKT, MEK/ERK signaling pathway inhibitor and Western blot technique to detect the regulation mechanism of cell signal pathway to YB-1 in pancreatic cancer cell line. The results showed that the ratio of yb-1 in pancreatic cancer was significantly higher than that in adjacent tissues. There was a positive correlation between YB-1 and clinical differentiation degree of pancreatic carcinoma. After using lentivirus to silence the expression of YB-1 in pancreatic cancer cell line, the proliferation of pancreatic carcinoma was significantly inhibited and G1 phase arrest appeared. The expression of GSK-3 尾 was upregulated in pancreatic cancer cell lines with lentivirus overexpression, and the proportion of cells in G 1 phase was significantly increased. After inhibiting YB-1, the level of GSK-3 尾 mRNA in pancreatic cancer cells decreased significantly. The change of protein expression level was consistent with that of mRNA. The expression of GSK-3 尾 mRNA and protein was up-regulated after overexpression of YB-1. The proliferation of pancreatic cancer cell line YB-1 overexpression was inhibited after GSK-3 尾 specific inhibitor was used to treat the overexpression of YB-1. The expression level of Cyclin D1 F1E 1 protein was consistent with that of cell proliferation. Using the inhibitors of PI3K/AKT and MEK/ERK signaling pathway, it was found that YB-1 was regulated by these two signaling pathways at the same time. Among them, PI3K/AKT is regulated at the transcription level and MEK / ERK is regulated at the translation level. Conclusion the overexpression of PI3K/AKT is an important mechanism for the highly malignant biological behavior of pancreatic cancer. By regulating the expression of GSK-3 尾, it promotes the proliferation and progression of pancreatic cancer cells. YB-1 and GSK-3 尾 have the potential to become targets for the treatment of pancreatic cancer.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.9

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