本文關(guān)鍵詞： 胰腺導(dǎo)管腺癌 外顯子組測序 細(xì)胞分裂周期相關(guān)蛋白2 預(yù)后 化療耐藥 腫瘤微環(huán)境　出處：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景及目的胰腺癌是常見的消化系統(tǒng)惡性腫瘤。在美國人群惡性腫瘤死因里排在第4位,在我國人群惡性腫瘤死因里排在前十位。胰腺癌惡性程度高、早期發(fā)現(xiàn)困難、進(jìn)展迅速、手術(shù)切除率低、對放化療不敏感、預(yù)后差,5年生存率約8%。隨著基因組學(xué)技術(shù)的發(fā)展,通過組學(xué)研究,在胰腺癌中尋找新的特異的早期診斷指標(biāo)來提高早期診斷率,并且明確胰腺癌化療敏感性的分子靶點(diǎn)從而提高化療效果,具有重要的臨床意義。本課題組前期工作中,通過激光捕獲顯微切割純化胰腺導(dǎo)管腺癌細(xì)胞進(jìn)行外顯子組測序,篩選得到胰腺癌與癌旁組織中表達(dá)的差異基因CDCA2。細(xì)胞分裂周期相關(guān)蛋白2 (cell division cycle associated 2, CDCA2)是蛋白磷酸酶1的一個(gè)結(jié)合亞基,主要作用是負(fù)責(zé)在有絲分裂后期,幫助蛋白磷酸酶與染色質(zhì)的結(jié)合,協(xié)助細(xì)胞完成有絲分裂的過程。在乳腺癌、口腔鱗癌、卵巢癌中均發(fā)現(xiàn)其蛋白表達(dá)較高,并與腫瘤惡性程度相關(guān),其機(jī)理可能因其促進(jìn)細(xì)胞周期進(jìn)展和抑制細(xì)胞凋亡發(fā)揮促進(jìn)腫瘤生長的作用。但目前尚未發(fā)現(xiàn)關(guān)于CDCA2在胰腺導(dǎo)管腺癌中作用的相關(guān)研究報(bào)道。另一方面,近年來對腫瘤微環(huán)境的研究逐漸深入,對于腫瘤微環(huán)境中的一大類細(xì)胞即巨噬細(xì)胞,通過其表型改變影響了癌細(xì)胞的功能,這一現(xiàn)象已在乳腺癌中得到了證實(shí)。在胰腺癌中同樣也觀察到巨噬細(xì)胞影響了癌細(xì)胞的侵襲能力。而對腫瘤相關(guān)巨噬細(xì)胞在作用于癌細(xì)胞的過程中,癌細(xì)胞自身凋亡能力發(fā)生變化的過程尚未明確。本研究通過對胰腺導(dǎo)管腺癌的外顯子組測序,篩選出表達(dá)差異基因CDCA2,進(jìn)行系列的功能調(diào)控驗(yàn)證,了解CDCA2蛋白表達(dá)與胰腺癌患者臨床病理特征及預(yù)后的關(guān)系,探索CDCA2與胰腺癌化療敏感性之間存在的聯(lián)系。同時(shí)探索腫瘤相關(guān)巨噬細(xì)胞與癌細(xì)胞作用的過程中,CDCA2發(fā)生的改變。研究內(nèi)容1.研究胰腺癌組織中CDCA2蛋白的表達(dá)情況及與胰腺癌患者臨床病理特征及預(yù)后的關(guān)系；2.研究CDCA2的RNA表達(dá)量與胰腺癌患者臨床病理特征及預(yù)后的關(guān)系；3.研究胰腺癌細(xì)胞株細(xì)胞中CDCA2的表達(dá)及對胰腺癌細(xì)胞株細(xì)胞增殖、凋亡能力的影響及相關(guān)機(jī)制；4.探索腫瘤相關(guān)巨噬細(xì)胞與胰腺癌的作用過程中,CDCA2表達(dá)的改變。方法：1.應(yīng)用免疫組織化學(xué)染色(immunohistochemistry,IHC)技術(shù)檢測手術(shù)切除胰腺癌組織標(biāo)本中CDCA2蛋白的表達(dá)情況,分析CDCA2蛋白表達(dá)與胰腺癌患者臨床病理特征及預(yù)后的關(guān)系。2.利用TCGA數(shù)據(jù)庫的公開數(shù)據(jù),分析胰腺癌患者CDCA2的RNA表達(dá)水平與臨床病理特征及預(yù)后的關(guān)系。3.利用western blot技術(shù)檢測各胰腺癌細(xì)胞株細(xì)胞中CDCA2的基礎(chǔ)表達(dá)情況。應(yīng)用siRNA轉(zhuǎn)染技術(shù)敲低其中高表達(dá)細(xì)胞株細(xì)胞中的CDCA2表達(dá),并設(shè)置相應(yīng)的對照組。CCK8法檢測CDCA2對胰腺癌細(xì)胞增殖能力的影響,利用流式細(xì)胞術(shù)檢測細(xì)胞周期及凋亡,利用Transwell法檢測CDCA2對胰腺癌細(xì)胞侵襲及遷移能力的影響,western blot檢測CDCA2影響胰腺癌細(xì)胞表型的作用機(jī)制。4.體外將U937細(xì)胞誘導(dǎo)分化為巨噬細(xì)胞M0(M(?))和M2,利用共培養(yǎng)體系分別將兩種巨噬細(xì)胞與胰腺癌細(xì)胞共培養(yǎng),CCK8法檢測胰腺癌細(xì)胞的增殖能力,利用流式細(xì)胞術(shù)檢測胰腺癌細(xì)胞的凋亡,western blot檢測巨噬細(xì)胞作用與胰腺癌細(xì)胞過程中CDCA2蛋白表達(dá)的改變。結(jié)果：1. CDCA2蛋白在胰腺導(dǎo)管腺癌組織中的表達(dá)水平高于癌旁組織,p0.001。CDCA2蛋白表達(dá)水平的高低、不同N分期所對應(yīng)的胰腺導(dǎo)管腺癌患者總生存期有顯著性差異(p0.05),是影響預(yù)后的危險(xiǎn)因素。多因素分析中發(fā)現(xiàn)CDCA2蛋白表達(dá)不是影響胰腺癌患者預(yù)后的獨(dú)立危險(xiǎn)因素。2. CDCA2的RNA表達(dá)水平與胰腺癌患者的腫瘤T分期(p=0.010),腫瘤分期(p=0.038)相關(guān)。CDCA2的RNA表達(dá)水平與患者的生存期之間的關(guān)系不存在統(tǒng)計(jì)學(xué)差異,p=0.067。3.敲低CDCA2的表達(dá)顯著降低胰腺癌細(xì)胞株BxPC-3與T3M-4細(xì)胞的增殖能力,但卻出現(xiàn)了減少細(xì)胞自身凋亡的現(xiàn)象,而對吉西他濱誘導(dǎo)的細(xì)胞凋亡是增加的。Western blot顯示CDCA2通過影響細(xì)胞周期蛋白CyclinD1、CDK4、CDK6的表達(dá),調(diào)控細(xì)胞周期,影響細(xì)胞的增殖能力,通過影響cleaved caspase-3和cleaved RARP的表達(dá)影響細(xì)胞的凋亡。4.M2巨噬細(xì)胞與胰腺癌細(xì)胞株BxPC-3和T3M-4細(xì)胞共培養(yǎng)后,明顯降低了胰腺癌細(xì)胞的增殖能力,也促進(jìn)了細(xì)胞凋亡。共培養(yǎng)過程中CDCA2的表達(dá)被下調(diào)了。結(jié)論：CDCA2基因是我國胰腺導(dǎo)管腺癌患者中較常見的突變基因。CDCA2蛋白在胰腺導(dǎo)管腺癌中的表達(dá)明顯高于癌旁組織。CDCA2蛋白表達(dá)高的患者預(yù)后較差。在胰腺癌細(xì)胞中,CDCA2可調(diào)控細(xì)胞周期,促進(jìn)胰腺癌細(xì)胞的增殖,同時(shí)影響細(xì)胞的凋亡能力。下調(diào)胰腺癌細(xì)胞中CDCA2的表達(dá),可以增加吉西他濱誘導(dǎo)的細(xì)胞凋亡。腫瘤相關(guān)巨噬細(xì)胞影響胰腺癌細(xì)胞的增殖和凋亡能力,在共培養(yǎng)過程中,下調(diào)了CDCA2的表達(dá)。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.9

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