【作者】 周英姿；

【导师】 桂律； 范卫； 史继敏； 叶宣光；

【作者基本信息】 复旦大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 背景：Fox(forkhead)蛋白家族是在进化上具有一个螺旋-转角-螺旋基序、3α螺旋和2个大环形成翼状(winged)结构保守氨基酸序列的DNA结合结构域的一种转录因子。其中Fox转录因子家族FOXO(Forkhead box, classO)包括FOXO1A、FOXO3A、MLLT7(FOX04)等成员。FOXO主要通过转录调控和信号传导途径在动物的生长发育、细胞分化、代谢、寿命、炎症和免疫等方面起重要作用。大量的哺乳动物体外实验及人类肿瘤组织研究证实磷酸化的FOXO1A(pFOXO1A)与多种肿瘤的发生、发展相关,是一种肿瘤相关基因。在国内,FOXO1A在糖尿病、横纹肌肉瘤等疾病中的表达见文献报道,但在胃癌中的表达及生物学意义的研究还鲜有述及。连环蛋白(catenin)是一组具有相似结构的细胞内糖蛋白家族,包括α-、β-、γ-和p12ctn四大类。它们能与钙粘蛋白胞内肽段结合,参与细胞粘附、生长、增殖和细胞转归过程,并在细胞信号转导中起重要作用。β-catenin的主要功能包括：同E-cadherin一起组成E-cadherin/β-catenin复合体,在细胞间粘附过程中起关键作用；协同转录因子参与Wnt信号通道转导。β-catenin现已被确认为是一种癌基因,与多种肿瘤有关。细胞凋亡在肿瘤的发生和发展中有着重要的地位,凋亡异常是癌症的一个重要标志。凋亡异常会导致变异细胞易于遭受进一步的遗传损伤,并在肿瘤的发生过程中扮演了重要的角色。FLASH (Flice-associated huge protein)亦称为caspase8相关蛋白2,最初被定义为一种促凋亡蛋白,在Fas诱导的凋亡过程中起传递作用并参与TNF诱导的NF-kappaB激活、细胞存活和增值、细胞循环、细胞分化和分裂等。FLASH的这些功能对肿瘤发生起着重要的作用,但是目前为止对FLASH基因改变和FLASH蛋白在人类肿瘤中的表达还所知甚少。对FOXO1A、β-catenin及FLASH在胃癌中的表达进行研究,探讨它们在胃癌的发生、发展过程中的作用以及各基因间协同作用,以期为胃癌的生物学行为判断、诊断及基因治疗等方面提供一定的理论参考依据。目的：检测FOXO1A、β-catenin和FLASH在胃癌组织中的表达,分析它们的表达与各项临床病理指标及肿瘤分化程度间的关系。并对三种基因的表达情况进行关联性分析,探讨它们在胃癌的发生、发展过程中的作用。方法：应用免疫组化方法,对68例胃癌切除标本(带有癌旁正常组织)中FOXO1A、β-catenin及FLASH的基因表达情况进行检测。并进一步对其表达情况与患者的年龄、性别、肿块的位置、大小、淋巴结转移、浸润深度等临床病理参数及肿瘤分化程度的关系进行分析。结果：(1)FOXO1A在68例胃癌组织中的阳性表达率73.53%(50／68)。FOXO1A在胃癌中的表达与肿瘤的分化程度呈显著相关(P<0.05),男性患者表达率明显高于女性(P<0.05)。(2)β-catenin的阳性表达率为83.82%(57／68)。β-catenin在胃癌中的表达与肿瘤的分化程度呈显著相关(P<0.05)；β-catenin表达与性别显著相关,男性患者β-catenin表达率明显高于女性。(3)FLASH的阳性表达率为77.94%(53／68)。FLASH在胃癌中的表达只与肿瘤的分化程度相关(P<0.05),与患者的年龄、性别、肿块的位置、大小、淋巴结转移、浸润深度等无关。(4)FOXO1A表达和β-catenin表达有显著关联性(P=0.0391),FLASH和β-catenin表达间无显著关联,FLASH和FOXO1A亦无显著关联。(4)在癌旁组织中,三种基因的表达主要在上皮内肿瘤区域,表达率为100%且呈较强阳性表达。在正常黏膜区域,三种基因不表达或呈极低表达,按照判定标准可视为阴性。结论：(1)FOXO1A在胃癌肿瘤发生过程中是一种早期频发分子事件。(2)β-catenin的表达与胃癌的分化程度密切相关,在胃癌的肿瘤分化过程中发挥了一定的作用。(3)FOXO1A与β-catenin间通过Wnt信号系统相互关联,可能在胃癌的发生、发展过程中起协同作用。(4)FOXO1A,β-catenin及FLASH在胃癌的诊治方面具有潜在的利用价值。更多还原

【Abstract】 Background:The winged helix/forkhead class of transcription factors is characterized by a monomeric DNA-binding domain, which folds into a variant of the helix-turn-helix motif and is made up of three a helices and two characteristic large loops, or wings. The FOXO(Forkheadbox, classO) is a subfamily of the forkhead transcription factor and consists of FOXOIA (FKHR:Forkhead in rhabdomyosarcoma, also known as FOXO1), FOXO3A.(FKHR-likel), and MLLT7(AFX:acute-lympho-cytic-leukaemia-1 fused gene from chromosome X, also known as FOXO4) plays an important role in the control of cell and organismal growth, development, differentiation, metabolism, longevity, inflammation and immunity. The function of FOXOIA has been most frequently evaluated in vivo in mammalian cancer cells and human cancer tissues, the expression of pFOXO1A has been correlated with tumorigenesis and tumor progression of many tumors. To date, the status of FOXOIA phosphorylation and its biological significance in human gastric cancer have not been reported in domestic literature. Catenin is an intracellular armadillo glycoprotein family, they are divided into 4 subfamilies:α-,β-,γ-and p120ctn. Catenin binds to the ctyplasmic tail of E-cadherin and forms an cell adhesion complex (Cadherin-Catenin-Complex, CCC), the complex plays an important role in maintaining epithelial cell adhesion. Some catenins(β-,γ-and p120ctn) are also crucial in cell signaling, they play an important role in cell proliferation and apoptosis. Now as an oncogene,β-catenin is related to many different types of tumors. Apoptosis is the most fundamental form of physiologic cell death that is essential for tissue homeostasis. Deregulation of apoptosis is one of the hallmarks of cancer. Failure of apoptosis could allow survival of transformed cells that are prone to undergo further genetic damage and play an important role in the pathogenesis of tumors. Flice-associated huge protein (FLASH), also known as caspase8-associated protein 2, was first identified as a pro-apoptotic protein involved in Fas-induced apoptosis. In addition to the apoptosis function, diverse biologic roles of FLASH, including activates NF-kB by binding with TRAF2 in tumor necrosis factor signaling, cell-cycle progression and cell division, and is required for histone transcription. Although such functions are important in tumor pathogenesis, little is known about the alterations of FLASH gene and FLASH protein expression in human cancers. In this study, we did research on expressions of FOXO1A,β-catenin and FLASH in gastric carcinoma(GC), and furthermore analyzed the role and relationship of them in GC, hoping that could supply some theoretical references for the tumor’s biological behavior, diagnosis and targeted therapy.Objective:To investigate the expression of FOXO1A,β-catenin and FLASH in human gastric carcinoma and their possible implications.Methods:FOXO1A,β-catenin and FLASH expression were examined by immunohistochemical staining method in 68 cases of human gastric cancer with human normal gastric tissue. Further analysis were performed to make sure it’s relationship with tumor clinico-pathological features, like age, gender, tumor location, size, lymphatic metastasis and grading and so on.Results:(1) By immunohistochemistry, FOXOIA protein expression in cancer cells was detected positively in 50 gastric carcinoma tissues(73.53%)。Significant associations between FOXOIA expression and grading and gender were found (P <0.05). (2) Of 68 tumors 57 were consideredβ-catenin positive(83.82%). The expression ofβ-catenin was inversely correction with tumor grading (P<0.05) Moreover gender was also involved. (3) In gastric carcinomas, the expression of FLASH was 77.94%(53/68). There were no significant associations between FLASH and age, tumor location, tumor size, depth of penetration or lymphatic metastasis, its expression was only significantly associated with tumor grading. (4) The expression of FOXO1A was correlated with that ofβ-catenin (P=0.0391), there was no significant correlation between FOXO1A and FLASH (or between FLASH andβ-catenin). (5) In non-neoplastic gastric mucosa, the expression of pFOXO1A was observed primarily in cells in intraepithelial neoplasia and presents intensive positively. Whereas in epithelial cells of normal gastric mucosa were shown as no or very weak intensity.Conclusions:(1) The expression of FOXOIA is a frequent and early event in gastric tumorigenesis. (2) The expression ofβ-catenin was closely related to the tumor grading of gastric carcinoma, and could play a certain role in it. (3) The expression ofβ-catenin may activate the expression of target gene FOXO1A through the Wnt signaling pathway, which plays a synergic role in carcinogenesis and progression of gastric carcinoma.(4) Our data suggest that the expression of the three genes may have potential application of diagnosis and targeted therapy in gastric carinoma.更多还原