本文選題：胰腺癌　切入點：P2X7受體　出處：《山東大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:胰腺癌作為一種消化道惡性腫瘤,其五年生存率不足5%。胰腺癌早期或術后發(fā)生的鄰近及遠處器官轉移是導致其手術切除率低及臨床高死亡率的主要原因,所以研究調控胰腺癌發(fā)展及侵襲轉移的關鍵分子,對胰腺癌臨床治療及預后有重大意義。P2X7受體蛋白作為一種以ATP為配體的門控型離子通道受體,其在多種腫瘤組織中高表達,并參與了腫瘤的發(fā)生、發(fā)展及凋亡等多種病理生理過程。我們本次的實驗主要探索P2X7受體蛋白在胰腺癌中的表達及臨床意義,以及其調節(jié)胰腺癌侵襲遷移的潛在機制。方法:1.應用Western blot實驗和qRT-PCR實驗分析人胰腺癌組織、癌旁組織標本中P2X7受體蛋白的表達情況,并應用免疫組織化學方法分析人胰腺癌組織中P2X7受體蛋白的表達與胰腺癌臨床病理特點之間的關系。2.選取正常胰腺細胞系HPDE6-C7及常見胰腺癌細胞系SW1990、PANC-1、BxPC-3、MiaPaCa-2及PANC02.03,應用Western blot實驗和細胞免疫熒光實驗檢測上述細胞系中P2X7受體蛋白的表達情況;并篩選出P2X7受體蛋白高表達的胰腺癌細胞株,分別設置對照組,P2X7受體激動劑BzATP(2000μM)處理組,P2X7 受體激動劑 BzATP(200μM)+抑制劑 A740003(20μM)處理組,應用 Transwell實驗、細胞劃痕實驗檢測上述處理后所選細胞株侵襲遷移能力變化。3.為進一步探索P2X7受體蛋白對胰腺癌細胞侵襲遷移的潛在機制,選取P2X7受體蛋白高表達細胞株,分別設置對照組,P2X7受體激動劑BzATP(200μM)處理組,P2X7受體激動劑BzATP(2000μM)+抑制劑A740003(20μM)處理組,應用Western blot實驗檢測上述處理后該細胞株中侵襲遷移相關蛋白MMP2、MMP9、E-cadherin、Vimentin的表達情況和對AKT信號通路關鍵蛋白AKT蛋白磷酸化激活的影響。結果:1.Western blot實驗和qRT-PCR實驗結果顯示P2X7受體蛋白在胰腺癌組織中高表達,在癌旁組織中低表達;且P2X7受體蛋白表達水平與胰腺癌的臨床腫瘤分化程度(低分化)及淋巴結轉移呈顯著性相關(P0.05)。2.Western blot實驗和細胞免疫熒光實驗結果顯示P2X7受體在胰腺癌細胞中高表達,并且選取P2X7受體高表達的PANC-1細胞進行Transwell實驗、細胞劃痕實驗結果顯示,P2X7受體蛋白的激活可促進PANC-1細胞的侵襲遷移。3.免疫印跡實驗結果顯示,PANC-1細胞中P2X7受體蛋白激活后,可下調E-cadherin蛋白表達,上調Vimentin、MMP2、MMP9蛋白的表達,且使pAKT蛋白的表達升高且呈時間依賴性。結論:1.P2X7受體蛋白在胰腺癌中高表達,并與胰腺癌的分化程度及淋巴結轉移顯著相關。2.P2X7受體蛋可能通過AKT信號通路調節(jié)E-cadherin、Vimentin、MMP2、MMP9蛋白的表達進而影響胰腺癌的侵襲及遷移。意義:1.研究了 P2X7受體蛋白與胰腺癌臨床病理特征之間的關系,為進一步探索P2X7受體蛋白在胰腺癌發(fā)展中的作用提供相關的實驗理論依據。2.P2X7受體蛋白可介導胰腺癌的侵襲及遷移,為胰腺癌的分子靶向治療研究提供了一個潛在的的靶點。
[Abstract]:Objective: as a malignant tumor of digestive tract, the 5-year survival rate of pancreatic cancer is less than 5 years. The metastasis of adjacent and distant organs in the early stage or after operation of pancreatic cancer is the main cause of low resection rate and high clinical mortality. Therefore, the study of key molecules regulating the development, invasion and metastasis of pancreatic cancer is of great significance for the clinical treatment and prognosis of pancreatic cancer. P2X7 receptor protein, as a gated ion channel receptor with ATP as a ligand, is highly expressed in various tumor tissues. Our experiment mainly explored the expression and clinical significance of P2X7 receptor protein in pancreatic cancer. Methods: 1. The expression of P2X7 receptor protein in human pancreatic carcinoma tissues and adjacent tissues was analyzed by Western blot assay and qRT-PCR assay. The relationship between the expression of P2X7 receptor protein and the clinicopathological characteristics of pancreatic carcinoma was analyzed by immunohistochemical method. 2.The normal pancreatic cell line HPDE6-C7, the common pancreatic cancer cell lines SW1990, PANC-1, BxPC-3, MiaPaCa-2 and PANC02.03were selected, and the Western blot was applied to analyze the relationship between the expression of P2X7 receptor protein and the clinicopathological characteristics of pancreatic carcinoma. The expression of P2X7 receptor protein in the above cell lines was detected by immunofluorescence assay. A pancreatic cancer cell line with high expression of P2X7 receptor protein was screened. The control group treated with P2X7 receptor agonist BzATP(2000 渭 M was treated with P2X7 receptor agonist BzATP(200 渭 M (A740003 渭 M), and the treatment group was treated with Transwell. In order to further explore the potential mechanism of P2X7 receptor protein on invasion and migration of pancreatic cancer cells, a cell line with high expression of P2X7 receptor protein was selected. The control group treated with P2X7 receptor agonist BzATP(200 渭 M and the P2X7 agonist BzATP(2000 渭 M inhibitor A740003 (20 渭 M) were treated with P2X7 receptor agonist A740003 (20 渭 M). Western blot assay was used to detect the expression of invasion and migration associated protein MMP2MMP9 E-cadherin vimentin and its effect on the activation of phosphorylation of AKT protein, a key protein in the AKT signaling pathway. Results 1. The results of Western blot and qRT-PCR experiments showed P2X7. The receptor protein is highly expressed in pancreatic carcinoma. Low expression in paracancerous tissues; The expression of P2X7 receptor protein was significantly correlated with the clinical differentiation (low differentiation) and lymph node metastasis of pancreatic carcinoma. The results of Western blot assay and cellular immunofluorescence assay showed that P2X7 receptor was highly expressed in pancreatic cancer cells. The results of Transwell assay showed that the activation of P2X7 receptor protein could promote the invasion and migration of PANC-1 cells. Western blot showed that P2X7 receptor protein was activated in PANC-1 cells. It can down-regulate the expression of E-cadherin protein, up-regulate the expression of VimentintMMP2MMP9 protein, and increase the expression of pAKT protein in a time-dependent manner. Conclusion: 1. P2X7 receptor protein is highly expressed in pancreatic carcinoma. P2X7 receptor egg may regulate the expression of E-cadherin Vimentinnus MMP2MMP9 protein through AKT signaling pathway, and then affect the invasion and migration of pancreatic carcinoma. Significance: 1. To study P2X7 receptor protein and pancreatic pancreas. The relationship between clinicopathological features of cancer, In order to further explore the role of P2X7 receptor protein in the development of pancreatic cancer to provide relevant experimental theoretical basis .2.P2X7 receptor protein can mediate the invasion and migration of pancreatic cancer, which provides a potential target for the molecular targeted therapy of pancreatic cancer.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.9

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