垂体腺瘤发病机理的基因表达谱分析及B10细胞在放疗后垂体腺瘤组织中变化的研究

发布时间：2018-05-18 17:47

本文选题：垂体腺瘤 + 白细胞介素-6　；参考：《山东大学》2016年博士论文

【摘要】：垂体是重要的内分泌器官,通过分泌几个重要的激素:催乳素(PRL),生长激素(GH),促肾上腺皮质激素(ACTH),促甲状腺激素(TSH)等,在机体中发挥着至关重要的作用。垂体前叶通过调节靶腺激素分泌,参与组织器官的正常发育和生长。垂体的异常严重扰乱机体的代谢平衡,不同的器官会有不同程度的异常表现。垂体腺瘤是一种生长在垂体前叶的特殊颅内肿瘤,它不只具有一般肿瘤的特征还具有导致内分泌紊乱的特点。垂体腺瘤大约占到脑肿瘤的10～20%,为多见的单克隆抗体起源肿瘤,在颅内肿瘤中发病率接近脑膜瘤,排在胶质瘤和脑膜瘤后面居第三位。大部分的垂体腺瘤为良性,只有小部分具有侵袭性,其中0.1～0.2%最终癌变,垂体腺癌的恶性程度与其预后密切相关。垂体腺瘤分为功能性腺瘤和非功能性腺瘤两类,其临床表现有包括占位效应和内分泌损害两方面。非功能性腺瘤多于功能性腺瘤,在功能性腺瘤当中,发病率最高为泌乳素腺瘤(PRL型),其下依次为生长激素腺瘤(GH型)、促肾上腺皮质激素腺瘤(ACTH型)、促卵泡和黄体生成素腺瘤(FSHLH)。功能性腺瘤的临床症状主要由激素分泌紊乱导致的。两类腺瘤在生长增大到一定程度时均可引起相应的占位效应。垂体腺瘤由一系列的垂体关键基因突变引起,这些基因包括蛋白激酶C(PKC)、p16、GADD45γ等。由于垂体腺瘤临床表现的变异性和肿瘤的生长不可预测性,引发了众多研究者的的持续关注。以往的研究表明,垂体腺瘤可以从不同的角度对身体的发育和生长产生负面的影响。垂体腺瘤引起的过量激素分泌会产生一系列代谢紊乱及脏器损伤。另一方面,由于肿瘤的压迫,引起其他激素分泌的降低,会导致靶腺功能的下降。目前,对垂体腺瘤的化疗及手术治疗的研究已经有大量的报道。已有的分子生物学研究表明,某些调控因子的基因和蛋白表达在垂体腺瘤中起着至关重要的作用。研究发现p53对垂体腺瘤的发生有着抑制作用,这种抑制作用可以被多形性腺瘤基因(pleomorphic adenoma gene-like 1,PLAGL1)与 RPRM,P21及佛波醇12肉豆蔻酸13醋酸酯诱导蛋白(phorbol-12-myristate-13-acetate-induced Protein 1,PMAIP1)的联合作用所破坏。众所周知GADD45β的过度表达可以通过激活细胞凋亡抑制因子抑制肿瘤的生长,这表明Gadd45β可能对垂体腺瘤也有潜在的抑制作用。垂体腺瘤可导致多种基因表达水平的升高或下降,其中大部分的变化也显示对肿瘤发生的调控作用。虽然一些研究报告涉及到了垂体腺瘤对潜在靶基因的影响,到目前为止仍没有满意的解决方法系统性的通过对高通量数据分析的方法,对基因表达库进行研究以分析垂体腺瘤引起的基因和蛋白表达差异。本研究旨在通过基因表达谱分析将垂体腺瘤与正常垂体对照研究,以探讨相关基因表达的类型和变化。之后,通过建立蛋白质相互作用(PPI)的差异表达基因网络(DEGs),分析了垂体腺瘤差异性表达基因的影响以及不同的差异性蛋白质之间的相互作用。研究的目的是通过在正常垂体及垂体腺瘤筛选差异表达基因及其蛋白产物,分析它们之间的相互作用,以研究垂体腺瘤的发病机理。通过搜集公共功能基因组学数据存储库(public functional genomics data repository)的基因表达谱(gene expression profiling),筛选出正常垂体及垂体腺瘤之间的差异表达基因(differential expressed genes,DEGs)。基因表达谱数据集GSE26966下载于功能性基因组数据库GEO。在纳入研究的23个样品中,9个样本取自正常垂体及14个样本取自垂体腺瘤。所有探针集的注释信息由Afffymetrix 人类基因组 u133a2.0 阵列(Affmetrix Human Genome U133 Plus 2.0 Array)提供。在数据处理和差异表达分析时,把CEL文件中探针水平的数据通过affy包系统的稳健多元阵列平均函数(robust multi-array average,RMA)转换成探针表达式值矩阵,并通过该数据集的芯片平台R/Bioconductor注解包将编码转变为基因名称。由于一个基因有许多相应的探针,最终将所有探针的表达式值的平均值通过计算(归一化)对应同一个基因的表达值。垂体腺瘤和正常垂体对比的差异表达基因(DEGs)通过R软件limma包的贝叶斯线性模型识别,只有 log fold change(LFC)值1.5 和错误发现率(false discovery rate,FDR)为校正的P0.05的基因才被选择为差异表达基因(DEGs)。为了确保筛选的差异表达基因(DEGs)可以很好的体现样本特征,本研究对差异表达基因(DEGs)进行了聚类分析并且绘制了聚类图。上调和下调的差异性表达基因功能则是通过对基因本体(gene ontology GO)的功能富集分析进行进一步的研究。之后,基因序列被映射到数据库,构建成上调和下调的差异性表达基因的蛋白质相互作用(protein-protein interactionPPI)网络。研究发现下调差异表达基因(DEGs)的PPI网络表现出相对集中的特点,网络中一些节点蛋白如EGR1,STAT3,JUNB和FOS都是癌症中常见的转录因子。相对的,上调差异表达基因(DEGs)的PPI网络表现出稀疏的状态。通过这两个PPI网络之间的比较,证明下调基因在垂体腺瘤中起着主要作用。最后,对下调的差异表达基因的蛋白质相互作用(PPI)网络的功能模块进行分析。本研究在正常垂体和垂体腺瘤样本间一共筛选出211个上调和413个下调差异表达基因。通过GO富集分析PPI网络建立,发现下调的差异表达基因与免疫反应、激素调节和细胞增殖等功能相关。上调的差异性表达基因与阳离子转运功能相关。从下调的差异表达基因的PPI网络获得五个模块。其中四个具有明显的生物学作用,其中的转录因子,如IL-6,STAT3,BCL6,EGR1,POU1F1,JunB和Fos是这些功能模块的核心节点。本研究通过对正常垂体和垂体腺瘤基因表达谱和PPI网络的筛选成功地找到差异表达基因及其相关的蛋白质。结果表明,激素和免疫相关基因的低表达促进垂体腺瘤的发生。低表达的IL6和STAT3在垂体腺瘤的免疫异常中扮演了关键的角色。同时,POU1F1低表达导致垂体激素分泌的减少,是垂体腺瘤的重要诱因。垂体腺瘤理想治疗目标为调整患者激素水平至正常范围,消除瘤体对周围组织的压迫,缓解瘤体在颅内诱发的不良症状及体征等。目前临床上垂体腺瘤常用的治疗方案为手术切除和放射治疗,但二者在实际临床应用中均存在一定的局限性。手术切除作为垂体腺瘤临床治疗的首选方案,虽然能有效缓解瘤体对周围组织的压迫,下调患者激素水平,但若瘤体切除不完全或肿瘤已出现周围组织侵犯,可导致手术的风险性增加,诱发诸多并发症并易发生术后复发。放射治疗主要用于术后复发、残留及不耐受或拒绝手术患者,作为垂体腺瘤治疗的二线方案,放射治疗可抑制肿瘤的生长,恢复患者激素分泌水平,但治疗周期较长,且有研究显示放射治疗可诱发垂体功能减退,损伤颅内神经细胞,严重时还可导致恶性肿瘤的发生。因此结合患者的临床病理特征,权衡利弊选择合理的治疗方案对垂体腺瘤患者预后改善有着重要意义。研究证实肿瘤的发生发展是一个复杂、多步骤的连续过程,免疫逃逸作为肿瘤恶化进展的关键,目前普遍认为其与肿瘤周围微环境的改变有关[1]。正常状态下,免疫系统的淋巴细胞可通过抗原识别完成对恶变细胞和自体细胞的有效区分,但在肿瘤细胞中,它一方面可通过降低或沉默自身免疫原性来躲避免疫系统的抗原识别;另一方面它可激活免疫抑制细胞如调节性T细胞(Treg)和调节性B细胞(Breg),诱导分泌免疫抑制细胞因子如TGF-β和IL-10[2],在肿瘤病灶组织周围形成免疫抑制网络最终实现免疫逃逸。对Treg细胞在肿瘤发生发展中的作用国内外学者已展开的大量的研究,Breg细胞作为另一大类具有特殊免疫抑制功能细胞,以往研究多集中在自身免疫性疾病、移植免疫耐受、感染与炎症反应等方面,但伴随研究深入,发现Breg细胞也参与促肿瘤的生长及转移[3,4]。包括皮肤良性肿瘤的发生、抑制T淋巴细胞的抗肿瘤效应、抑制肝癌细胞的凋亡、增强肝癌细胞的增殖和迁移活性。临床研究也证实在卵巢癌、胃癌、肺癌、胰腺癌、乳腺癌等恶性实体瘤中可见Bregs细胞的浸润并与肿瘤微环境的免疫抑制及肿瘤的恶性侵袭密切相关。鉴于Bregs在肿瘤进展中的重要作用,针对Bregs的靶向药物研究成为目前的关注热点,尤其是肿瘤局部浸润Bregs,通过直接干预Bregs活化数量消除其负性调控,或间接抑制Bregs分泌的细胞因子恢复免疫监视功能发挥抗肿瘤效应,成为目前肿瘤治疗研究的新型靶向。B10细胞作为Bregs的亚型,研究显示这一类含量稀少表型特殊的B细胞在肿瘤逃逸过程中有着重要作用,包括参与CLL免疫抑制调控[11]、胰腺癌的进展过程[12]。研究证实放射治疗过程中肿瘤细胞的凋亡可导致肿瘤抗原的释放进而激活机体的先天免疫信号[13],并伴随肿瘤微环境免疫抑制的减弱[14]。这也提示我们在垂体腺瘤反射治疗过程中,是否同样存在B10细胞的变化,因此围绕垂体腺瘤放疗患者组织内B10细胞变化我们展开了相关研究,收集复发垂体腺瘤患者48例,23例术前接受放射治疗,25例未接受治疗,患者经手术摘除垂体腺瘤后,通过检测垂体腺瘤患者组织CD19+CD1d+CD5+和B10细胞亚群比例,测定垂体腺瘤患者组织miR-98和HDAC1 mRNA表达,明确放射治疗过程中垂体腺瘤患者组织B10细胞变化特点。研究发现放射治疗可下调垂体腺瘤患者组织中B10细胞及其亚群CD19+CD1d+CD5+和CD19+CD24+CD38+数量及分布频率;放射治疗垂体腺瘤患者组织中miR-98 mRNA表达显著上调,提示放射治疗可促进miR-98表达,miR-98表达上调可抑制IL-10转录,进而影响B10细胞免疫抑制功能。
[Abstract]:The pituitary gland is an important endocrine organ, which plays a vital role in the body by secreting a few important hormones, such as PRL, GH, ACTH, and TSH. The anterior pituitary is involved in the normal development and growth of the tissues and organs by regulating the secretion of the target gland hormone. The pituitary adenoma is a special intracranial tumor that grows in the anterior pituitary. It is not only characteristic of the general tumor, but also has the characteristics of endocrine disorder. The pituitary adenoma accounts for about 10 to 20% of the brain tumor, which is a common monoclonal antibody. The incidence of tumor origin is close to meningioma in intracranial tumors and third in glioma and meningioma. Most pituitary adenomas are benign, only a small part is invasive, of which 0.1 to 0.2% are eventually cancerous, and the malignancy of pituitary adenocarcinoma is closely related to the prognosis. Pituitary adenomas are divided into functional adenomas and nonfunctional glands. There are two types of tumor, including two aspects of space occupying effect and endocrine damage. Nonfunctional adenomas are more than functional adenomas. Among functional adenomas, the highest incidence is prolactin adenoma (type PRL), which is followed by growth hormone adenoma (type GH), adrenocorticotropic hormone adenoma (ACTH type), follicle promoting and luteinizing adenoma (FSH LH. The clinical symptoms of functional adenomas are mainly caused by hormonal disorder. The two types of adenomas may cause the corresponding occupying effect when the growth is increased to a certain extent. Pituitary adenomas are caused by a series of mutations in the key pituitary genes, including protein kinase C (PKC), p16, GADD45 gamma, and so on. The unpredictability of the growth of the heterosexual and tumor growth has led to the continuous attention of many researchers. Previous studies have shown that pituitary adenomas can have a negative impact on the development and growth of the body from different angles. The excessive hormone secretion caused by pituitary adenomas may produce a series of metabolic disorders and organ damage. On the other hand, the swelling is due to swelling. The pressure of the tumor, causing the decrease of other hormone secretion, will lead to the decline of the function of the target gland. At present, there have been a lot of reports on the chemotherapy and surgical treatment of pituitary adenomas. Molecular biology studies have shown that the gene and protein expression of some regulatory factors play a vital role in pituitary adenomas. Research has found that p53 It has an inhibitory effect on the occurrence of pituitary adenomas, which can be destroyed by the combination of the pleomorphic adenoma gene-like 1, PLAGL1 and RPRM, P21 and the 12 myristic acid 13 acetate induced protein (phorbol-12-myristate-13-acetate-induced Protein 1, PMAIP1). Overexpression can inhibit tumor growth by activating the inhibitory factor of apoptosis, which suggests that Gadd45 beta may also have a potential inhibitory effect on pituitary adenomas. Pituitary adenomas may lead to a rise or decline in a variety of gene expression levels, and most of the changes also indicate the regulatory role of the tumor. The effect of pituitary adenoma on potential target genes has not been satisfactorily solved so far. The gene and protein expression differences caused by pituitary adenomas are analyzed by the method of high throughput data analysis to analyze the difference in gene and protein expression caused by pituitary adenomas. Normal pituitary control study to explore the types and changes of related gene expression. After that, the differential expression gene network (DEGs) of protein interaction (PPI) was established to analyze the influence of differentially expressed genes in pituitary adenomas and the interaction between different differential proteins. The purpose of this study was to pass through the normal pituitary and drooping. Body adenoma screening differentially expressed genes and their protein products to analyze the interaction between them in order to study the pathogenesis of pituitary adenomas. By collecting the gene expression profiles (gene expression profiling) of the public functional genomics data repository (gene expression profiling), the normal pituitary gland and the pituitary gland were screened. The differential expression gene (differential expressed genes, DEGs). The gene expression profile data set GSE26966 was downloaded from the functional genomic database GEO. in the 23 samples taken into the study. 9 samples were taken from the normal pituitary and 14 samples were taken from the pituitary adenoma. The annotation information of all the probe sets was derived from the Afffymetrix human genome u133a2. The.0 array (Affmetrix Human Genome U133 Plus 2 Array) is provided. In data processing and differential expression analysis, the probe level data in the CEL file is converted to the probe expression value matrix through the robust multivariate array average function of the Affy packet system (robust multi-array average,) and through the chip platform of the data set The uctor annotated packet transforms the encoding into a gene name. As a gene has a number of corresponding probes, the average value of the expression value of all probes is finally calculated (normalized) to correspond to the expression value of the same gene. The differential expression gene (DEGs) of the pituitary adenoma and the normal pituitary (DEGs) is used by the Bayesian linear model of the R software package. Identification, only the log fold change (LFC) value 1.5 and the error discovery rate (false discovery rate, FDR) are selected as the differentially expressed genes (DEGs). In order to ensure that the selected differentially expressed genes (DEGs) can well reflect the sample characteristics, the differentially expressed genes (DEGs) are cluster analysis and plotted in this study. The function of differentially expressed genes between up and down is further studied by the functional enrichment analysis of gene ontology GO. After that, the gene sequence is mapped to the database to construct the protein interaction (protein-protein interactionPPI) network of up and down differentially expressed genes (protein-protein interactionPPI). The study found that the PPI network that down regulated differentially expressed genes (DEGs) showed a relatively concentrated characteristic. Some of the nodes in the network, such as EGR1, STAT3, JUNB and FOS, were common transcription factors in cancer. Relative, the PPI network up regulating the differential expression gene (DEGs) showed a sparse state. By comparison of these two PPI networks Down regulated genes play a major role in pituitary adenomas. Finally, the functional modules of the protein interaction (PPI) network of down regulated differentially expressed genes were analyzed. In this study, 211 up-regulated and 413 down regulated differentially expressed genes were screened in normal pituitary and pituitary adenoma samples. The PPI network was established by GO enrichment analysis. The down regulated differentially expressed genes were related to the functions of the immune response, hormone regulation and cell proliferation. The up-regulated differentially expressed genes were related to the cation transport function. Five modules were obtained from the PPI network of down regulated differentially expressed genes. Four of them had obvious biological use, such as the transcription factors, such as IL-6, STAT3, BCL6, E. GR1, POU1F1, JunB and Fos are the core nodes of these functional modules. This study successfully found differentially expressed genes and related proteins by screening gene expression profiles and PPI networks of normal pituitary and pituitary adenomas. The results showed that the low expression of hormone and immune related genes promoted the occurrence of pituitary adenomas. Low expression of IL6 and STAT. 3 plays a key role in the immune abnormality of pituitary adenoma. At the same time, the low expression of POU1F1 leads to the decrease of pituitary hormone secretion. It is an important cause of pituitary adenoma. The ideal treatment of pituitary adenoma is to adjust the level of the hormone to the normal range, eliminate the oppression of the tumor body to the surrounding tissue and alleviate the adverse symptoms induced by the tumor in the intracranial. At present, surgical resection and radiotherapy are commonly used in the clinical treatment of pituitary adenomas, but there are some limitations in the actual clinical application of the two. Surgical excision is the first choice for clinical treatment of pituitary adenomas, although it can effectively alleviate the compression of the surrounding tissue and reduce the level of the hormone in the patient, but if the tumor is tumor An incomplete resection of the body or an invasion of the surrounding tissue may lead to an increase in the risk of surgery, a number of complications and postoperative recurrence. Radiation therapy is mainly used for postoperative recurrence, residual and intolerance or rejection of the operation, as a second line of pituitary adenoma treatment. Radiation therapy can inhibit the growth of the tumor and restore the patient. The level of hormone secretion is long, but the treatment cycle is longer, and there are studies showing that radiation therapy can induce hypophysis dysfunction and injury of intracranial nerve cells, and it can also lead to malignant tumor. Therefore, combining the clinicopathological features of the patients, choosing a reasonable treatment scheme to weigh the advantages and disadvantages is important to improve the prognosis of pituitary adenoma patients. Research confirms that the development of tumor is a complex, multi step process. Immune escape is the key to the progression of cancer. It is generally believed that it is related to the change of the microenvironment around the tumor, which is related to the normal state of [1].. The lymphocyte of the immune system can be used to complete the effective cells and autologous cells through the antigen recognition. But in tumor cells, it can avoid the antigen recognition of the immune system by reducing or silent autoimmunity, on the other hand, it activates the immunosuppressive cells such as regulatory T cells (Treg) and regulatory B cells (Breg), inducing secretory cytokines such as TGF- beta and IL-10[2], around the tumor tissue. The formation of immunosuppressive networks finally realizes immune escape. The role of Treg cells in the development of tumors has been extensively studied by domestic and foreign scholars. Breg cells have special immunosuppressive functions as another major class. Previous studies focused on autoimmune diseases, transplantation immune tolerance, infection and inflammation, and so on. However, with the further study, it is found that Breg cells also participate in the growth of tumor and the metastasis of [3,4]. including benign tumor of the skin, inhibit the anti-tumor effect of T lymphocyte, inhibit the apoptosis of hepatoma cells, enhance the proliferation and migration activity of the hepatoma cells. The clinical study also confirmed that the malignant tumor, gastric cancer, lung cancer, pancreatic cancer, and breast cancer are malignant. The infiltration of Bregs cells in solid tumors is closely related to the immunosuppression of the tumor microenvironment and the malignant invasion of the tumor. In view of the important role of Bregs in the progression of the tumor, the research on targeted drugs for Bregs has become the focus of attention, especially the local infiltration of Bregs in the tumor, and to eliminate the negative effects of the number of Bregs activated by direct intervention in the number of Bregs. The regulation, or the indirect inhibition of the cytokines secreted by Bregs to restore the immune surveillance function to play an antitumor effect, has become a new target.B10 cell for cancer therapy as a subtype of Bregs. The study shows that this kind of rare B cells with rare phenotypes have an important role in the process of tumor escape, including participation in the CLL immune suppression. [11], the progress of pancreatic cancer [12]. research confirms that the apoptosis of tumor cells in the course of radiation therapy can lead to the release of tumor antigen and then activate the organism's innate immune signal [13], and with the decrease of [14]. in the tumor microenvironment immunity inhibition, it also suggests that we also have B10 cells in the process of pituitary adenoma counter shoot treatment. The changes of B10 cells in the tissue of patients with pituitary adenoma were studied, 48 cases of recurrent pituitary adenomas were collected, 23 patients received radiotherapy before operation, 25 cases were untreated. After surgical removal of pituitary adenomas, the proportion of CD19+CD1d+CD5+ and B10 cell subgroups in the pituitary adenoma was detected by detecting the pituitary adenoma. The expression of miR-98 and HDAC1 mRNA in the tissue of pituitary adenoma was determined and the characteristics of B10 cell changes in the tissue of pituitary adenoma patients were determined. The study found that radiation therapy could reduce the number and frequency of B10 cells and their subgroups CD19+CD1d+CD5+ and CD19+CD24+CD38+ in the tissues of pituitary adenomas; radiation therapy for pituitary adenoma patients group. The expression of miR-98 mRNA was significantly up-regulated, suggesting that radiotherapy can promote the expression of miR-98. Up regulation of miR-98 can inhibit the transcription of IL-10 and further affect B10.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R736.4

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