胰腺导管腺癌中细胞分裂周期相关蛋白2的作用及机制研究

发布时间：2018-02-28 05:15

本文关键词： 胰腺导管腺癌外显子组测序细胞分裂周期相关蛋白2 预后化疗耐药肿瘤微环境　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：研究背景及目的胰腺癌是常见的消化系统恶性肿瘤。在美国人群恶性肿瘤死因里排在第4位,在我国人群恶性肿瘤死因里排在前十位。胰腺癌恶性程度高、早期发现困难、进展迅速、手术切除率低、对放化疗不敏感、预后差,5年生存率约8%。随着基因组学技术的发展,通过组学研究,在胰腺癌中寻找新的特异的早期诊断指标来提高早期诊断率,并且明确胰腺癌化疗敏感性的分子靶点从而提高化疗效果,具有重要的临床意义。本课题组前期工作中,通过激光捕获显微切割纯化胰腺导管腺癌细胞进行外显子组测序,筛选得到胰腺癌与癌旁组织中表达的差异基因CDCA2。细胞分裂周期相关蛋白2 (cell division cycle associated 2, CDCA2)是蛋白磷酸酶1的一个结合亚基,主要作用是负责在有丝分裂后期,帮助蛋白磷酸酶与染色质的结合,协助细胞完成有丝分裂的过程。在乳腺癌、口腔鳞癌、卵巢癌中均发现其蛋白表达较高,并与肿瘤恶性程度相关,其机理可能因其促进细胞周期进展和抑制细胞凋亡发挥促进肿瘤生长的作用。但目前尚未发现关于CDCA2在胰腺导管腺癌中作用的相关研究报道。另一方面,近年来对肿瘤微环境的研究逐渐深入,对于肿瘤微环境中的一大类细胞即巨噬细胞,通过其表型改变影响了癌细胞的功能,这一现象已在乳腺癌中得到了证实。在胰腺癌中同样也观察到巨噬细胞影响了癌细胞的侵袭能力。而对肿瘤相关巨噬细胞在作用于癌细胞的过程中,癌细胞自身凋亡能力发生变化的过程尚未明确。本研究通过对胰腺导管腺癌的外显子组测序,筛选出表达差异基因CDCA2,进行系列的功能调控验证,了解CDCA2蛋白表达与胰腺癌患者临床病理特征及预后的关系,探索CDCA2与胰腺癌化疗敏感性之间存在的联系。同时探索肿瘤相关巨噬细胞与癌细胞作用的过程中,CDCA2发生的改变。研究内容1.研究胰腺癌组织中CDCA2蛋白的表达情况及与胰腺癌患者临床病理特征及预后的关系；2.研究CDCA2的RNA表达量与胰腺癌患者临床病理特征及预后的关系；3.研究胰腺癌细胞株细胞中CDCA2的表达及对胰腺癌细胞株细胞增殖、凋亡能力的影响及相关机制；4.探索肿瘤相关巨噬细胞与胰腺癌的作用过程中,CDCA2表达的改变。方法：1.应用免疫组织化学染色(immunohistochemistry,IHC)技术检测手术切除胰腺癌组织标本中CDCA2蛋白的表达情况,分析CDCA2蛋白表达与胰腺癌患者临床病理特征及预后的关系。2.利用TCGA数据库的公开数据,分析胰腺癌患者CDCA2的RNA表达水平与临床病理特征及预后的关系。3.利用western blot技术检测各胰腺癌细胞株细胞中CDCA2的基础表达情况。应用siRNA转染技术敲低其中高表达细胞株细胞中的CDCA2表达,并设置相应的对照组。CCK8法检测CDCA2对胰腺癌细胞增殖能力的影响,利用流式细胞术检测细胞周期及凋亡,利用Transwell法检测CDCA2对胰腺癌细胞侵袭及迁移能力的影响,western blot检测CDCA2影响胰腺癌细胞表型的作用机制。4.体外将U937细胞诱导分化为巨噬细胞M0(M(?))和M2,利用共培养体系分别将两种巨噬细胞与胰腺癌细胞共培养,CCK8法检测胰腺癌细胞的增殖能力,利用流式细胞术检测胰腺癌细胞的凋亡,western blot检测巨噬细胞作用与胰腺癌细胞过程中CDCA2蛋白表达的改变。结果：1. CDCA2蛋白在胰腺导管腺癌组织中的表达水平高于癌旁组织,p0.001。CDCA2蛋白表达水平的高低、不同N分期所对应的胰腺导管腺癌患者总生存期有显著性差异(p0.05),是影响预后的危险因素。多因素分析中发现CDCA2蛋白表达不是影响胰腺癌患者预后的独立危险因素。2. CDCA2的RNA表达水平与胰腺癌患者的肿瘤T分期(p=0.010),肿瘤分期(p=0.038)相关。CDCA2的RNA表达水平与患者的生存期之间的关系不存在统计学差异,p=0.067。3.敲低CDCA2的表达显著降低胰腺癌细胞株BxPC-3与T3M-4细胞的增殖能力,但却出现了减少细胞自身凋亡的现象,而对吉西他滨诱导的细胞凋亡是增加的。Western blot显示CDCA2通过影响细胞周期蛋白CyclinD1、CDK4、CDK6的表达,调控细胞周期,影响细胞的增殖能力,通过影响cleaved caspase-3和cleaved RARP的表达影响细胞的凋亡。4.M2巨噬细胞与胰腺癌细胞株BxPC-3和T3M-4细胞共培养后,明显降低了胰腺癌细胞的增殖能力,也促进了细胞凋亡。共培养过程中CDCA2的表达被下调了。结论：CDCA2基因是我国胰腺导管腺癌患者中较常见的突变基因。CDCA2蛋白在胰腺导管腺癌中的表达明显高于癌旁组织。CDCA2蛋白表达高的患者预后较差。在胰腺癌细胞中,CDCA2可调控细胞周期,促进胰腺癌细胞的增殖,同时影响细胞的凋亡能力。下调胰腺癌细胞中CDCA2的表达,可以增加吉西他滨诱导的细胞凋亡。肿瘤相关巨噬细胞影响胰腺癌细胞的增殖和凋亡能力,在共培养过程中,下调了CDCA2的表达。
[Abstract]:Background and objective: pancreatic cancer is a common malignant tumor of digestive system in the United States. The population in malignant tumor death row in fourth, malignant tumor death in our population ranked in the top ten. Pancreatic cancer, early detection, rapid progress, the resection rate is low, is not sensitive to chemotherapy. Poor prognosis, 5 years survival rate is about 8%. with the development of genomics, by the study group, looking for new specific indicators of early diagnosis in pancreatic cancer to improve the early diagnosis and specific molecular target sensitivity of pancreatic cancer chemotherapy so as to improve the effect of chemotherapy and has important clinical significance. The preparatory work the research group, using laser capture microdissection and purification of pancreatic ductal adenocarcinoma cells by exome sequencing, differential expression screening of pancreatic cancer and adjacent tissues by gene CDCA2. cell cycle related protein 2 (cell Division cycle associated 2, CDCA2) is a binding subunit of protein phosphatase 1, the main role is responsible for late in mitosis, with the help of protein phosphatase and chromatin, assist in cell mitosis. In oral squamous cell carcinoma, breast cancer, ovarian cancer was found in the protein expression is higher, and associated with the degree of malignancy, and its mechanism may be due to the promotion of cell cycle progression and inhibition of apoptosis play a role in promoting tumor growth. But has not yet found the relevant research reports about the role of CDCA2 in pancreatic ductal adenocarcinoma. On the other hand, in recent years, the research on the tumor microenvironment gradually, for a large class of cells in the tumor microenvironment that macrophages through its phenotypic changes affect cancer cell function, this phenomenon has been confirmed in breast cancer. In pancreatic cancer was also observed in macrophages. Rang of cancer cell invasion ability. And the process of tumor associated macrophages in cancer cells, cancer cell apoptosis ability change is not yet clear. Through the study of pancreatic ductal adenocarcinoma exome sequencing, screening of differentially expressed genes in CDCA2, functional verification regulation of series, understand the relationship between CDCA2 protein expression and clinical pathological characteristics and prognosis of patients with pancreatic cancer, explore between CDCA2 and pancreatic cancer chemotherapy sensitivity. At the same time to explore the process of tumor associated macrophages and the role of CDCA2 in cancer cells, occurs. The change of expression of CDCA2 protein contents of 1. pancreatic cancer tissue and its relation with clinical pathology the characteristics and prognosis of patients with pancreatic cancer; the expression of CDCA2 RNA 2. to study the relationship between amount of pancreatic cancer patients with clinical pathological characteristics and prognosis; 3. of pancreatic cancer cell line cells The expression of CDCA2 on cell proliferation and apoptosis of pancreatic cancer cell lines, the ability and mechanism of action; 4. to explore the process of tumor associated macrophages and pancreatic cancer, the expression of CDCA2. Methods: 1. immunohistochemical staining (immunohistochemistry, IHC) expression detection technology of surgical resection of CDCA2 protein in pancreatic cancer tissue the analysis of CDCA2 protein expression of.2. and clinical pathological characteristics and prognosis of patients with pancreatic cancer using publicly available data from the TCGA database,.3. CDCA2 analysis of the relationship between the expression level of RNA in patients with pancreatic cancer and clinical pathological characteristics and prognosis of Western by using blot technique to detect the expression of CDCA2 in pancreatic carcinoma cell lines by siRNA cells. Transfected cell lines CDCA2 on which high and low expression, and set the corresponding control group CDCA2.CCK8 method for detection of pancreatic cancer cells Effect of proliferation, cell cycle and apoptosis were detected by flow cytometry, detected by Transwell CDCA2 on pancreatic cancer cell invasion and migration ability, Western blot CDCA2.4. in vitro detection mechanism of phenotype of pancreatic cancer cells will induce the differentiation of U937 cells into macrophage M0 (M (?)) and M2, using the the co culture system were two kinds of macrophages and pancreatic cancer cells were cultured to detect pancreatic cancer cells CCK8 proliferation, apoptosis of pancreatic cancer cells were detected by flow cytometry, the expression of CDCA2 protein in Western blot detection process of macrophage function and pancreatic cancer cells change. Results: the expression level of CDCA2 protein in pancreatic duct 1. adenocarcinoma was higher than that in the adjacent tissues, the expression of p0.001.CDCA2 protein in the level of overall survival in patients with pancreatic ductal adenocarcinoma with different N stages corresponding to the significant differences (p0. 05), is the risk factors influencing the prognosis. The expression of CDCA2 protein is not the independent risk factors affecting the prognosis of patients with pancreatic cancer.2. CDCA2 and RNA expression level in patients with pancreatic cancer T staging of the tumor found in multivariate analysis (p=0.010), tumor stage (p=0.038) related to the relationship between.CDCA2 RNA expression and survival of patients there is no statistically significant difference, p=0.067.3. knockdown CDCA2 protein significantly decreased in pancreatic cancer cell line BxPC-3 and T3M-4 cell proliferation, but appeared to reduce cell apoptosis phenomenon, and the cell apoptosis induced by gemcitabine was increased.Western blot CDCA2 by affecting the expression of cyclin CyclinD1, CDK4, CDK6 expression effect of cell cycle regulation, cell proliferation, cell apoptosis of.4.M2 macrophages and the effect of pancreatic cancer cells by affecting the expression of cleaved caspase-3 and cleaved RARP Strains of co cultured BxPC-3 and T3M-4 cells, significantly reduced pancreatic cancer cell proliferation, promote cell apoptosis. The expression of CDCA2 was reduced by co culture. Conclusion: CDCA2 gene is expressed in pancreatic ductal adenocarcinoma in patients with more common mutations of.CDCA2 gene in pancreatic ductal adenocarcinoma in adenocarcinoma the adjacent tissues with high expression of.CDCA2 protein of patients with poor prognosis. In pancreatic cancer cells, CDCA2 regulates cell cycle, promote pancreatic cancer cell proliferation, and apoptosis cells. Expression of CDCA2 in pancreatic cancer cells, can enhance apoptosis induced by gemcitabine. Tumor associated macrophage proliferation effect and apoptosis of pancreatic cancer cells, during coculture, down-regulation of CDCA2 expression.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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