【作者】 王伟民；

【导师】 李新钢；

【作者基本信息】 山东大学 ， 外科学（神经外）（专业学位）， 2016， 博士

【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(FSH&LH)。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P<0.05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。更多还原

【Abstract】 Pituitary gland,as an important endocrine organ,performs vital roles in organismal modulation through secreting several important hormones such as prolactin(PRL),growth hormone(GH),adrenocorticotropic hormone(ACTH),thyroid stimulating hormone(TSH).By regulating target glands hormone secretion,anterior pituitary participates in the development and normalization of tissues.Abnormality of pituitary tends to severely disrupt the body’s metabolic homeostasis,damaging various organs to different extents.Pituitary adenoma is a special intracranial tumor growing in the anterior pituitary which not just meets the characteristics of the tumor but also exhibits endocrine disorder.Pituitary adenoma accounts for about 10～20%of brain tumors,with a monoclonal antibody of origin.The incidence rate in intracranial tumors has been close to in the back row of meningioma,It was only less than glioma and meningioma.Most of the pituitary adenomas are benign,only a small part of aggressive,of which 0.1～0.2%eventually carcinogenesis,There is a close relationship between the prognosis of pituitary adenocarcinoma and its malignant degree.Pituitary adenomas were divided into functional adenoma and non functional adenoma,its clinical manifestations include occupying effect and endocrine lesions in two aspects,non functional adenoma has a higher incidence rate than functional adenoma.Incidence rate from high to low are:prolactinomas(PRL type),growth hormone adenoma(type GH),adrenocorticotropic hormone producing adenoma(ACTH),follicle stimulating hormone and luteinizing hormone adenoma(FSH&LH).The clinical symptoms of functional adenoma is mainly composed of hormone secretion disorder caused.Two adenomas can be found the occupying effect When the tumor has grown big enough.Pituitary adenoma caused by mutations in a series of pituitary key genes such as protein kinase C(PKC),P16,and growth arrest and DNA damage-45y(GADD45γ)has provoked continued attentions among endocrinologists due to its variability in clinical presentation and unpredictability of tumor growth.As showed in previous studies,pituitary adenoma can affect the body development in negative ways from various perspectives.Excess hormone secretion in pituitary caused by pituitary adenoma produce a series of metabolic disorder and visceral injury.On the contrary,other hormones present down-regulation because of compression of tumor which subsequently results in function decline of target glands.Currently,a wealth of studies has been conducted on the surgical treatment and chemotherapy of pituitary adenoma.Studies showed that both gene and protein expressions of certain regulatory factors present vital roles in the pituitary adenoma.It was found that P53 inhibits the generation of pituitary adenoma and this function could be restrained by pleomorphic adenoma gene-like 1(PLAGL1)under the joint action with RPRM,P21 and phorbol-12-myristate-13-acetate-induced Protein 1(PMAIP1).It is well acknowledged that over expression of growth arrest and DNA damage-45β(GADD45-beta)could inhibit tumor growth through activating apoptosis inhibiting factors,which indicating that GADD45-beta may also serve a potential inhibition to pituitary adenoma.Pituitary adenoma can lead to a variety of gene expression level raise or decline,most changes of which also shows regulatory effects on tumorigenesis.Although some studies were reported concerning effects of pituitary adenoma on potential target genes,there has no effective detection method through the flux way to systematically detect the gene and protein differential expressions caused by pituitary adenoma.The present study aims to investigate types and changes of gene expression in pituitary adenoma compared to normal pituitary through gene expression profiling.Subsequently,by establishing PPI(protein-protein interaction)network of differentially expressed genes(DEGs),we analyzed the effects of differential proteins on pituitary adenoma and the interactions among diverse differential proteins.We aim to study the pathogenesis of pituitary adenoma through screening the differential expressed genes(DEGs)and proteins between normal pituitary and pituitary adenoma,and analyzing the interactions among them.After acquisitioning gene expression profiling from public functional genomics data repository,DEGs were screened between normal pituitary and pituitary adenoma.The gene expression profile GSE26966 was downloaded from a public functional genomics data repository GEO.Among the total 23 samples been investigated,9 and 14 samples were respectively from normal pituitary and pituitary adenoma.The annotation information of all probes sets was provided by Affymetrix Human Genome U133A2.0 Array.For data processing and differential expression analysis,we converted the probe-level data in CEL files into expression value of probe matrix by RMA(robust multi-array average)in Affy package,and transferred the serial numbers into gene names by platform R/Bioconductor note package.Finally,as one gene having many corresponding probes,we calculated the average value of all expression value of probes as the expression value of a single gene.The Bayesian linear model of limma package in R software was used to identify DEGs in pituitary adenoma compared to normal pituitary.Only genes with the |log fold change| value larger than 1.5 and false discovery rate(FDR)less than 0.05 were selected as DEGs.In order to ensure the screened DEGs can well characterize the samples,clustering analysis and dendrogram for DEGs were established respectively.Up and down-regulated DEGs were further identified through gene ontology(GO)functional enrichment analysis.Subsequently,the DEGs were mapped to string database and then protein-protein interaction(PPI)networks of the up and down-regulated DEGs were constructed.The PPI network of the down-regulated DEGs presented a characteristic of centralization,and some node proteins of the network such as EGR1,STAT3,JUNB and FOS were the common transcription factors in cancer.Relatively,the PPI network of up-regulated DEGs showed a sparse status.Through the comparison between these two PPI networks,we found that down-regulated genes played a main role in pituitary adenoma.Thus,we focused on the analysis of functional modules in PPI network of down-regulated DEGs.Finally,functional modules of PPI network of down-regulated DEGs were analyzed.Totally,211 up-regulated and 413 down-regulated DEGs were screened between normal and pituitary adenomas samples.Down-regulated DEGs were associated with functions like immune response,hormone regulation and cell proliferation.Up-regulated genes were related to cation transport function.Five modules were acquired from the PPI network of the down-regulated DEGs.Transcription factors,such as STAT3,IL6,Bc16,EGR1,POU1F1,JUNB and FOS were the core nodes in functional modules.In summary,we succeeded to find the DEGs and proteins through screening gene expression profiling and PPI networks.Results of the present study indicated that low expression of hormone and immune related genes facilitated the occurrence of pituitary adenoma.Low expression of IL6 and STAT3 played pivotal roles in the dysimmunity of pituitary adenoma.Meanwhile,low expression of POU1F1 contributed to the pituitary hormone secretion decline.Pituitary adenomas are tumors occurred in the pituitary gland,which consists of benign type,malignant type and invasive type.If the tumor size is larger than 1 cm,the tumors are designated pituitary macroadenomas.In spite of the pathological types,pituitary macroadenomas often cause hyperpituitarism or hypopituitarism and a series of subsequent diseases.Although some pituitary microadenomas(less than 1 cm in size)may be remained untreatment if the tumors do not cause clinical symptoms,most pituitary macroadenomas require surgical intervention.Tumor tolerance indicates a condition that immune effector cells do not attack tumor cells;tumors thus grow out of control.The underlying mechanism of tumor tolerance is suggested that tumor specific regulatory T cells or/and tumor specific regulatory B cells develop in tumor tissue to inhibit the tumor specific effector cell function.Transforming growth factor(TGF)-β and interleukin(IL)-10 are two major immune regulatory molecules;by releasing which the tumor specific immune regulatory cells interfere with immune effector cell functions.Radiotherapy is one of the major remedies in tumor treatment.The therapeutic efficacy needs to be further improved.Probiotics can facilitate immune regulation.This study tests a hypothesis that radiotherapy inhibits interleukin(IL)-10-producing B cells(B10 cells)in pituitary adenomas.Patients with recurrentpituitary adenomas were treated with radiotherapy prior to surgery.B10 cells in tumor tissue were analyzed after surgical removal.The results showed that the frequency of B10 cells in pituitary adenoma tissue was significantly less in those treated with radiotherapy.The B10 cells from pituitary adenoma without irradiation showed high levels of HDAC1 and low levels of miR-98.Exposure B cells to radiation increased miR-98 expression;the latter inhibits IL-10 expression.A conclusioncould be drawn from the results:radiation suppresses IL-10 expression in B cells in pituitary adenomas via up regulation of miR-98 expression.更多还原