基于基因表达谱的乳腺癌外周血与肿瘤局部免疫微环境关联性研究

发布时间：2018-04-22 23:06

本文选题：乳腺癌 + 外周血　；参考：《电子科技大学》2016年博士论文

【摘要】：乳腺癌是常见的女性恶性肿瘤,具有异质性高的特点,即使细胞形态学和雌激素受体等细胞分子表型很相似的乳腺癌患者,也可能具有不同的预后表现,或对相同的治疗方案存在不同的反应。因此,除目前临床常用的指标外,需要更准确、更具临床实用性的预测因子,为乳腺癌个体化治疗提供依据。肿瘤的发展不仅由肿瘤细胞自身决定,也依赖于肿瘤细胞与间质之间复杂的相互作用。作为肿瘤免疫效应的执行场所,肿瘤微环境中包含了大量的免疫细胞,为肿瘤免疫逃逸提供条件,乳腺癌的免疫微环境是影响其发展和预后的重要因素之一。外周血是用于疾病诊断的最常用材料,被患者广泛接受,是无创性分子诊断的理想材料。而且,相比于肿瘤组织于手术中一次取样,忽略了微环境动态变化的特点,外周血可定期多次取样,便于监测。在乳腺癌发展的过程中,作为肿瘤微环境中免疫细胞的主要来源,关于乳腺癌患者外周血与肿瘤局部免疫微环境的关系,外周血是否能够反映肿瘤局部免疫微环境的变化,以及外周血来源的免疫微环境相关分子,目前尚无深入、系统的研究。肿瘤生物信息学是一种新兴的手段,将生物信息学的方法学、工具软件及数据库等应用于肿瘤的分子机制、新的肿瘤生物标志物和个体化治疗措施的研究中。本研究以基因表达谱数据为基础,联合生物信息学工具及文献挖掘手段,首先分析了良性和恶性乳腺肿瘤外周血单个核细胞的基因表达变化,评价外周血作为替代材料,用于评估乳腺癌患者的免疫状态可能性。然后,比较了乳腺癌外周血细胞与癌间质细胞差异基因表达的一致性,评价外周血与癌间质免疫微环境的关系。最后,筛选了乳腺癌组织与外周血细胞之间的交互作用分子,探讨乳腺癌组织对外周免疫系统可能的远程调节。本研究主要内容包括如下三部分:1、良性与恶性乳腺肿瘤外周血细胞基因差异表达研究。从GEO数据库中获取良性和恶性乳腺肿瘤患者外周血单个核细胞(PBMCs)mRNA表达谱。包括57例乳腺癌,37例良性乳腺肿瘤和31例健康人外周血标本。GEO2R工具分别筛选良性和恶性乳腺肿瘤相较于健康人群的外周血差异表达基因,DAVID工具富集基因功能和通路。STRING数据库构建差异表达基因蛋白产物相互作用的网络,筛选核心基因。良性和恶性乳腺肿瘤分别筛选到563和237个差异基因,两类患者外周血基因表达模式存在显著差异,其中乳腺癌差异基因功能侧重于免疫反应,涉及白细胞的激活、血管生成等生物学过程以及白细胞跨内皮迁移信号通路。IL-8、rhob、itgb1等为关键基因,可能在乳腺癌外周免疫激活中起关键作用。2、乳腺癌患者外周血与肿瘤间质免疫微环境关系研究。下载geo数据库中乳腺癌间质及外周血细胞mrna表达谱。乳腺癌间质样本来源于53例乳腺癌组织,31例匹配的癌旁正常组织。brb-arraytools预处理数据并筛选差异表达基因,david工具富集分析基因功能和通路。两组数据相同的差异表达基因仅为103个,占二者差异基因总数的2%。乳腺癌间质与外周血较正常对照的差异表达基因交叠数较少,但二者差异基因涉及的一致的生物学功能主要集中在免疫反应相关过程。进一步对差异表达的细胞因子的研究发现,pbmcs表达谱差异表达基因中共81个属于细胞因子、趋化因子及其互作基因(其中76个表达上调,5个下调)。乳腺癌间质表达谱中共103个属于细胞因子、趋化因子及其互作基因(其中55个表达上调,48个下调)。乳腺癌外周血细胞与间质的细胞因子相关基因的表达模式具有相似之处,其共同扰动的通路,均表现为免疫抑制和促肿瘤效应。3、乳腺癌组织与外周血的交互作用研究。下载geo数据库中乳腺癌间质、外周血细胞及乳腺癌上皮细胞mrna表达谱。乳腺癌上皮细胞样本来源于28例浸润性乳腺癌组织,5例正常乳腺组织。brb-arraytools预处理数据并筛选差异表达基因。利用uniprot、string、dlrp蛋白质相互作用数据库,对乳腺癌上皮细胞(间质细胞)差异表达上调的基因蛋白产物进行亚细胞定位,确定差异表达的分泌蛋白,及与其相互作用的乳腺癌pbmcs膜蛋白,作为分子对话中的受体。检索与受体相互作用的胞内下游蛋白质;使用cytoscape软件构建配体—受体—胞内下游蛋白质的相互作用网络。david工具对互作分子进行基因功能和通路富集。乳腺癌上皮细胞与pbmcs之间共得到101对相互作用分子,其中fn1、itgb1、flt1、comp、cxcl12为关键蛋白。乳腺癌间质细胞与pbmcs之间得到包括19个表达上调的乳腺癌间质细胞分泌蛋白(配体),26个pbmcs膜(受体),73个与pbmcs受体互作的胞内蛋白的交互作用网络,其中hub基因为itgb1,itgb3,itga2b,cxcr4。交互作用网络中的基因功能均主要涉及细胞粘附、细胞迁移、白细胞激活、免疫反应等生物学过程,参与的信号通路主要有细胞外基质—受体相互作用,细胞因子—细胞因子受体相互作用,趋化因子信号通路等。综上所述,利用基因表达谱芯片数据及生物信息学工具和方法,本论文探讨了乳腺癌患者外周血细胞的基因差异表达,分析了外周血与乳腺癌间质微环境在免疫反应方面的相似性。通过乳腺癌组织与外周血细胞可能的相互作用分子网络的分析,探讨这种相似性的分子机制。本研究发现,il-8、rhob、itgb1、fn1、flt1、comp、cxcl12、itgb3、itga2b、cxcr4等基因可能在乳腺癌患者以外周血为基础的免疫微环境的形成中发挥着重要作用。其中itgb1和cxcr4在乳腺癌外周血细胞显著上调,同时为乳腺癌细胞和间质细胞分泌蛋白共同的作用靶点,对其深入研究,干预肿瘤来源的分子与外周血细胞的远程相互作用,有可能改变外周免疫细胞向肿瘤局部的趋化,为乳腺癌的免疫治疗及预后监测提供新的思路。
[Abstract]:Breast cancer is a common female malignant tumor with high heterogeneity, and breast cancer patients with similar cell morphology and estrogen receptor phenotype are likely to have different prognosis or different responses to the same treatment. The more clinically practical predictors provide a basis for the individualized treatment of breast cancer. The development of the tumor is not only determined by the tumor cells themselves, but also depends on the complex interaction between the tumor cells and the interstitium. As the execution place of the tumor immune effect, a large number of immune cells are contained in the tumor microenvironment and escape from the tumor immunity. The immune microenvironment of breast cancer is one of the most important factors affecting the development and prognosis of breast cancer. Peripheral blood is the most commonly used material for diagnosis of disease. It is widely accepted by patients and is an ideal material for noninvasive molecular diagnosis. Moreover, compared to the one sampling in the operation of the tumor tissue, the characteristics of the dynamic changes in the microenvironment are ignored. Blood can be sampled regularly to facilitate monitoring. In the development of breast cancer, as the main source of immune cells in the tumor microenvironment, the relationship between peripheral blood and local immune microenvironment of the breast cancer patients, whether the peripheral blood can reflect the changes in the local immune microenvironment of the tumor, and the immune microenvironment of the peripheral blood There is no deep and systematic research on the biomarkers. Tumor bioinformatics is a new means to apply bioinformatics methodology, tool software and database to the molecular mechanism of tumor, new tumor biomarkers and individualized treatment. Informatics tools and literature mining methods first analyzed the gene expression changes in peripheral blood mononuclear cells of benign and malignant breast tumors, and evaluated the peripheral blood as an alternative material to evaluate the immune status of breast cancer patients. Then, the conformance of differentially expressed genes between the peripheral blood cells of breast cancer and the interstitial cells of breast cancer was compared. To evaluate the relationship between peripheral blood and intercellular immune microenvironment. Finally, the interaction molecules between breast cancer tissue and peripheral blood cells were screened and the possible remote regulation of the peripheral immune system of breast cancer tissues was explored. The main contents of this study include the following three parts: 1, the differential expression of the peripheral blood cell genes in benign and malignant breast tumors The expression profiles of peripheral blood mononuclear cells (PBMCs) mRNA in benign and malignant breast tumors were obtained from the GEO database, including 57 cases of breast cancer, 37 benign breast tumors and 31 healthy human peripheral blood samples by.GEO2R tool, respectively, to screen the peripheral blood differential expression genes of benign and malignant breast tumors compared to the Yu Jian Kang population, DAVID tool The gene function and pathway.STRING database constructed the network that differentially expressed gene protein products and screened the core genes. 563 and 237 differential genes were screened for benign and malignant breast tumors. There were significant differences in the expression patterns of peripheral blood gene in the two types of patients. The differential gene function of breast cancer focused on the immune response. The biological processes, such as leukocyte activation, angiogenesis and other biological processes, such as.IL-8, RhoB, itgb1, and so on, may play a key role in the peripheral immune activation of breast cancer, and the relationship between peripheral blood and tumor interstitial immune microenvironment in breast cancer patients. Downloading the interstitial and external breast cancer in the geo database. MRNA expression profiles of peripheral blood cells. The samples of breast cancer were derived from 53 breast cancer tissues, 31 matched paracancerous normal tissues with.Brb-arraytools preprocessing data and screening differentially expressed genes. The David tool enriched the function and pathway of the gene. The same differentially expressed genes in the two groups were only 103, accounting for the total number of differences in the total number of two 2%.. The differential expression of differentially expressed genes between the interstitial and peripheral blood of the breast cancer was less than that of the normal control, but the consistent biological function involved in the two differentially expressed genes was mainly concentrated in the immune response related process. Further research on the differentially expressed cytokines showed that the difference table of PBMCs expression spectrum reached 81 cytokine, and the chemokines were chemotactic. Gene expression profiles (76 up-regulated and 5 down-regulated). The expression profiles of mammary carcinoma were 103 cytokine, chemokines and their interaction genes (55 up and 48 down-regulation). The expression patterns of the peripheral blood cells and the cytokine related genes in breast cancer were similar, and their common disturbance pathway The interaction of immunosuppressive and tumor promoting effect.3 and the interaction of breast cancer tissue with peripheral blood. The mRNA expression profiles of breast cancer stroma, peripheral blood cells and breast cancer epithelial cells were downloaded in the geo database. The breast cancer epithelial cells were derived from 28 cases of invasive breast cancer and 5 normal breast tissues by.Brb-arraytools preconditioning Data and screening differential expression genes. Using UniProt, string, and DLRP protein interaction database, subcellular localization of gene protein products up regulated by differential expression of breast cancer epithelial cells (stromal cells), differentially expressed secretory proteins, and PBMCs membrane protein of breast cancer that are used for their interaction are used as the receptor in the molecular dialogue. To retrieve the intracellular downstream proteins interacting with the receptor, and use the Cytoscape software to construct the ligand receptor - intracellular downstream protein interaction network.David tool for gene function and pathway enrichment. There are 101 pairs of interacting molecules between breast cancer epithelial cells and PBMCs, of which FN1, itgb1, Flt1, comp, CXCL12 is a key protein. Between mammary cancer stromal cells and PBMCs, the intercellular protein (ligand), 26 PBMCs membranes (receptors), and 73 intercellular proteins interacting with PBMCs receptors, are obtained between mammary carcinoma cells and 19 up regulated breast cancer cells, in which the gene function of the hub gene is itgb1, ITGB3, itga2b, and cxcr4. interaction network It is mainly involved in biological processes such as cell adhesion, cell migration, leukocyte activation, and immune response. The signal pathways involved mainly include extracellular matrix receptor interaction, cytokine receptor interaction, chemokine signaling pathway and so on. In summary, the gene expression chip data and bioinformatics tools are used in summary. Methods, the gene differential expression of peripheral blood cells in breast cancer patients was discussed. The similarity between the peripheral blood and breast cancer microenvironment in the immune response was analyzed. The molecular mechanism of the possible interaction of breast cancer tissues and peripheral blood cells was analyzed to explore the molecular mechanism of this similarity. This study found that IL-8, Rho B, itgb1, FN1, Flt1, comp, CXCL12, ITGB3, itga2b, CXCR4 may play an important role in the formation of immune microenvironment based on the peripheral blood of breast cancer patients. Itgb1 and CXCR4 are up up in the peripheral blood cells of breast cancer, at the same time, for the common target of breast cancer cells and interstitial cells to secrete proteins. The long-range interaction between tumor sources and peripheral blood cells may change the chemotaxis of the peripheral immune cells to the tumor, and provide new ideas for the immunotherapy and prognosis monitoring of breast cancer.

【学位授予单位】：电子科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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