P2X7受体在胰腺癌中的表达及其对侵袭迁移影响的实验研究

发布时间：2018-03-06 06:22

本文选题：胰腺癌　切入点：P2X7受体　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:胰腺癌作为一种消化道恶性肿瘤,其五年生存率不足5%。胰腺癌早期或术后发生的邻近及远处器官转移是导致其手术切除率低及临床高死亡率的主要原因,所以研究调控胰腺癌发展及侵袭转移的关键分子,对胰腺癌临床治疗及预后有重大意义。P2X7受体蛋白作为一种以ATP为配体的门控型离子通道受体,其在多种肿瘤组织中高表达,并参与了肿瘤的发生、发展及凋亡等多种病理生理过程。我们本次的实验主要探索P2X7受体蛋白在胰腺癌中的表达及临床意义,以及其调节胰腺癌侵袭迁移的潜在机制。方法:1.应用Western blot实验和qRT-PCR实验分析人胰腺癌组织、癌旁组织标本中P2X7受体蛋白的表达情况,并应用免疫组织化学方法分析人胰腺癌组织中P2X7受体蛋白的表达与胰腺癌临床病理特点之间的关系。2.选取正常胰腺细胞系HPDE6-C7及常见胰腺癌细胞系SW1990、PANC-1、BxPC-3、MiaPaCa-2及PANC02.03,应用Western blot实验和细胞免疫荧光实验检测上述细胞系中P2X7受体蛋白的表达情况;并筛选出P2X7受体蛋白高表达的胰腺癌细胞株,分别设置对照组,P2X7受体激动剂BzATP(2000μM)处理组,P2X7 受体激动剂 BzATP(200μM)+抑制剂 A740003(20μM)处理组,应用 Transwell实验、细胞划痕实验检测上述处理后所选细胞株侵袭迁移能力变化。3.为进一步探索P2X7受体蛋白对胰腺癌细胞侵袭迁移的潜在机制,选取P2X7受体蛋白高表达细胞株,分别设置对照组,P2X7受体激动剂BzATP(200μM)处理组,P2X7受体激动剂BzATP(2000μM)+抑制剂A740003(20μM)处理组,应用Western blot实验检测上述处理后该细胞株中侵袭迁移相关蛋白MMP2、MMP9、E-cadherin、Vimentin的表达情况和对AKT信号通路关键蛋白AKT蛋白磷酸化激活的影响。结果:1.Western blot实验和qRT-PCR实验结果显示P2X7受体蛋白在胰腺癌组织中高表达,在癌旁组织中低表达;且P2X7受体蛋白表达水平与胰腺癌的临床肿瘤分化程度(低分化)及淋巴结转移呈显著性相关(P0.05)。2.Western blot实验和细胞免疫荧光实验结果显示P2X7受体在胰腺癌细胞中高表达,并且选取P2X7受体高表达的PANC-1细胞进行Transwell实验、细胞划痕实验结果显示,P2X7受体蛋白的激活可促进PANC-1细胞的侵袭迁移。3.免疫印迹实验结果显示,PANC-1细胞中P2X7受体蛋白激活后,可下调E-cadherin蛋白表达,上调Vimentin、MMP2、MMP9蛋白的表达,且使pAKT蛋白的表达升高且呈时间依赖性。结论:1.P2X7受体蛋白在胰腺癌中高表达,并与胰腺癌的分化程度及淋巴结转移显著相关。2.P2X7受体蛋可能通过AKT信号通路调节E-cadherin、Vimentin、MMP2、MMP9蛋白的表达进而影响胰腺癌的侵袭及迁移。意义:1.研究了 P2X7受体蛋白与胰腺癌临床病理特征之间的关系,为进一步探索P2X7受体蛋白在胰腺癌发展中的作用提供相关的实验理论依据。2.P2X7受体蛋白可介导胰腺癌的侵袭及迁移,为胰腺癌的分子靶向治疗研究提供了一个潜在的的靶点。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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