【作者】 赵大鹏；

【导师】 贾继辉；

【作者基本信息】 山东大学 ， 病原生物学， 2010， 博士

【摘要】 幽门螺杆菌(Helicobacter pylori, H. pylori)是革兰氏阴性、螺杆状的微需氧菌,可定植于胃粘膜上皮细胞,是慢性胃炎、消化道溃疡、胃粘膜相关淋巴组织(MALT)淋巴瘤及胃癌发生的危险因素,1994年世界卫生组织国际癌症研究机构(IARC)已将其列为Ⅰ类致癌因子。幽门螺杆菌的某些菌株包含有一段由27-33个基因编码的35-40kb cag致病岛(PAI). cagA基因是cag致病岛的重要组成部分之一,它编码了幽门螺杆菌的重要毒力蛋白CagA (120-140kDa)。CagA蛋白含有一段能够发生酪氨酸磷酸化的EPIYA基序,幽门螺杆菌黏附到胃粘膜上皮细胞以后,可通过四型分泌系统将毒力因子CagA蛋白注入上皮细胞。在cagA-positive幽门螺杆菌菌株的感染中,细菌源性的致癌因子CagA蛋白广泛的参与到细菌感染导致的胃组织损伤以及致癌的过程之中,但涉及的分子机制尚未完全明了。肿瘤发生的标志之一是细胞进入无序增殖状态,伴有一些参与到调节细胞周期进程、细胞的增殖、分化以及凋亡、衰老的重要细胞因子的表达紊乱。新鉴定的重要原癌蛋白CIP2A能通过抑制PP2A的活性来稳定MYC蛋白的表达并且促进肿瘤的形成。并且,在胃癌组织中CIP2A的表达量高于相对应的正常胃组织,其功能与胃癌细胞的增殖能力密切相关。而作为Btk酪氨酸激酶家族成员之一的BMX/Etk激酶的功能也涉及到对细胞增殖能力的促进以及对环境刺激的适应性调控。因此,阐明幽门螺杆菌感染和CIP2A之间的效应关系可以帮助我们更深入的理解幽门螺杆菌感染的致癌机理以及CIP2A在胃癌发生发展中的重要作用。胃癌的发生发展除了受到幽门螺杆菌的菌株特点影响外,还受到宿主的遗传易感性和环境因素的影响,与胃癌高发风险相关的环境因素之一就是高盐饮食。在人群的流行病学研究和动物模型中高盐饮食和胃癌的发病率之间的关系已经被充分证实。但是高盐饮食是通过何种机制增强了人群中胃癌的发病率尚未完全明了。因此,探讨高盐对细菌诱导的CIP2A表达增强效应可以帮助我们更深入的理解高盐饮食和幽门螺杆菌感染在胃癌发生发展过程中发挥协同作用的分子机制。而表没食子儿茶素没食子酸酯(EGCG)作为从天然植物绿茶中提取出来的单体成分(多酚类),其抑制肿瘤细胞增殖的能力已经通过动物实验模型、流行病学调查、以及病例对照研究等得到充分的证实。但EGCG抑制肿瘤发生发展的分子机制尚未完全阐明。因此,研究EGCG对细胞中CIP2A表达水平的影响可以为理解EGCG抑制肿瘤细胞增殖能力的分子机制提供更多的帮助。胃癌是世界范围内最常见的恶性肿瘤之一,中国是胃癌的高发国家,其致死率位居全球肿瘤相关致死率第二位。由于缺乏可靠的早期诊断标志,胃癌的预后情况并不乐观。促进胃癌发生发展的因素涉及生物致癌因素(幽门螺杆菌)和化学致癌因素(高盐和亚硝酸盐)的刺激；抑制胃癌发生发展的因素涉及多酚类(绿茶)和维生素类的摄入。肿瘤的分子诊断与预测,已成为近年来肿瘤研究的热点领域。而与胃癌发生发展相关的保护性和致癌性的因素是否都存在一个相同的作用靶点分子,至今尚未完全阐明。本研究的内容主要涉及幽门螺杆菌的感染以及环境因素中的高盐和EGCG的刺激对肿瘤细胞的增殖相关蛋白CIP2A表达的影响,旨在阐明CIP2A分子作为与胃癌发生发展相关的保护性和致癌性因素存在的共同作用靶点的依据。该课题主要研究内容及实验结果如下：一、幽门螺杆菌的感染能够促进CIP2A的表达上调幽门螺杆菌感染胃粘膜上皮细胞后,能激活一系列的信号途径,包括MEK／ERK途径,β-catenin途径,Src kinase途径,NF-κB途径,以及p38 MAP kinase途径。并且能够影响细胞内许多致癌因子的表达变化,例如影响MYC蛋白的稳定,影响β-catenin的核转移,以及影响细胞周期相关蛋白Cyclin Dl的表达。但是迄今为止,幽门螺杆菌的感染对原癌蛋白CIP2A表达的影响尚未明了。本研究通过实施幽门螺杆菌感染细胞以及CagA的表达质粒转染细胞的实验,以及应用了激酶抑制剂来验证哪几条信号通路参与了CagA诱导的CIP2A表达上调效应,此外实施了针对CIP2A的RNA干扰实验,以及干扰后幽门螺杆菌再次感染细胞的实验。通过western blot检测首次在细胞系中阐明了幽门螺杆菌的感染以及细菌源性的癌蛋白CagA诱导细胞的癌蛋白CIP2A表达上调的效应机理。CagA蛋白进入细胞后,激活了Src和MEK/ERK信号途径,并最终导致CIP2A的表达上调。本研究结果为深入理解幽门螺杆菌感染诱发胃癌的分子机制提供了有力的佐证。二、高盐刺激能够增强幽门螺杆菌感染所诱导的CIP2A表达上调本论文中第一部分的实验已经阐明了幽门螺杆菌的感染以及细菌源性的癌蛋白CagA诱导CIP2A表达上调的效应机理,为深入的理解幽门螺杆菌感染导致胃癌发生发展的分子机制提供了线索；同时,环境中高盐因素的刺激也是胃癌高发的危险因素之一,并且高盐饮食可能作为幽门螺杆菌感染导致胃癌的协同刺激因素存在。为了阐明高盐因素刺激幽门螺杆菌后对细菌的包括CagA在内的毒力因子表达的影响,以及高盐刺激后的幽门螺杆菌感染细胞是否比普通幽门螺杆菌感染细胞具有更强的诱导CIP2A表达上调的能力,我们设计了培养基中不同盐浓度刺激幽门螺杆菌后检测相关毒力基因表达量的实验以及高盐刺激后的幽门螺杆菌和普通的幽门螺杆菌分别感染细胞后对CIP2A表达量影响的western blot检测。实验结果表明环境中高盐因素的刺激能够诱导幽门螺杆菌毒力相关基因(cagA、vacA、tlpB、tip-alpha)的表达水平上调,经高盐刺激后毒力增强的细菌感染真核细胞后,可以增强细菌感染细胞所诱导的癌蛋白CIP2A表达上调效应,进而促进胃癌的发生发展；此外,通过对CIP2A分子的检测分析有助于理解高盐饮食和幽门螺杆菌感染在胃癌发生发展过程中发挥协同作用的分子机制。三、EGCG能通过抑制BMX和CIP2A来抑制肿瘤细胞的增殖EGCG抑制肿瘤发生发展的分子机制涉及到众多因子,包括：抑制癌蛋白Myc的表达、抑制基质金属蛋白酶(MMPs)的表达以及对P53和BCL-2等凋亡相关蛋白的影响等。而BMX/Etk酪氨酸激酶和CIP2A属于一类与肿瘤细胞增殖能力相关的重要细胞因子。而迄今为止,具有抗肿瘤活性的绿茶提取物EGCG和BMX激酶以及癌蛋白CIP2A之间的关系尚未实施探讨。因此,研究EGCG与BMX激酶以及癌蛋白CIP2A之间的关系,以及BMX和CIP2A之间的相互调节关系可以帮助我们更深入的理解EGCG在抑制肿瘤细胞增殖过程中发挥作用所涉及的分子机制。基于以上目的,设计了相关实验来验证EGCG作用细胞后对BMX和CIP2A蛋白表达量的影响；应用了真核表达质粒以及siRNA干扰技术来观察BMX和CIP2A之间的相互调控关系；此外,还进行了克隆形成实验来观察CIP2A在EGCG诱导的抑制肿瘤细胞增殖中的作用。实验结果证实了EGCG作用于细胞后可以导致细胞中CIP2A和BMX的表达水平降低,发现了BMX可以正向调控CIP2A的表达,并验证了EGCG发挥抑制肿瘤细胞增殖能力所可能涉及的分子机制。这就为更充分的理解EGCG抑制肿瘤细胞增殖能力的分子机理提供了更详实的依据。更多还原

【Abstract】 Helicobacter pylori (H. pylori) has been defined as a class I carcinogenic factor by WHO and its persistent colonization in stomach leads to an increased risk for peptic ulcers and gastric adenocarcinoma. Some H. pylori strains contain a 35-40 kb cag pathogenicity island (PAI) encoded by 27-33 genes. One constituent of the cag pathogenicity island (PAI) is called cagA which encodes a 120-140kDa CagA protein. CagA protein that has tyrosine phosphorylation (EPIYA) motifs is injected into the gastric epithelial cells by type IV secretory system (TFSS). cagA-positive H. pylori strains have a closer association with the progress of both peptic ulcers and gastric cancer than the cagA-negative strains according to epidemiological research. Within gastric epithelial cells, tyrosine phosphorylation occurs on the C-terminus of CagA by the Src family of tyrosine kinases. CagA interacts with many signal molecules and elicits a series of cellular events. Some changes related to cell morphology, cell scattering, cell proliferation and intercellular tight junctions have also been identified. According to these observations, bacterial oncoprotein CagA participates in gastric tissue injury caused by cagA-positive H. pylori infection.Unlimited cellular proliferation is a hallmark of tumors. Tumorigenesis is related to the disordered expression of some key factors which participate in the regulation of cell cycle progression, differentiation, apoptosis and senescence. Recent studies have reported that CIP2A or Cancerous Inhibitor of PP2A serves as an important oncoprotein. Protein phosphatase 2A (PP2A) can facilitate the proteolytic degradation of oncoprotein MYC and prevent malignant cell growth. More importantly, overexpression of CIP2A was observed in gastric cancer and its function was related to tumor cell proliferation. Btk family tyrosine kinase BMX was also involved in the promotion of tumor cell proliferation and regulation of cellular responses. Therefore, to elucidate the relationship between H. pylori infection and CIP2A expression could provide further evidence for understanding the mechanism of H. pylori carcinogenesis and CIP2A functions in gastric tumorigenesis.Gastric carcinogenesis is related to H. pylori infection, genetic susceptibility of hosts and environmental stimuli. High salt in food and drink is a very important risk factor which is involved in high incidence of gastric cancer. The close relationship between high salt in diet and gastric carcinogenic incidence has been proved through epidemic research and animal model. However, the mechanism of high salt intake-induced-gastric cancer high risk still remains unclear. For this reason, to investigate the enhancement effects of high salt on H. pylori infection-induced-CIP2A upregulation could help us to understand the molecular mechanism of synergistic effects between H. pylori infection and high salt stimuli in gastric carcinogenesis induction. As we all known, EGCG belongs to the most abundant polyphenol in green tea. The role of EGCG in the regulation of tumor cell proliferation has been verified in animal studies, human relevance and case-control reports. Up to now, the molecular mechanism of EGCG-inhibited-tumor cells proliferation still remains unclear. Therefore, to investigate the inhibitory effects of EGCG on CIP2A could provide further evidence for the understanding of the molecular mechanism of EGCG-induced-tumor inhibition.Gastric cancer is one of the most common tumors in worldwide and has a high incidence in China. The prognosis of gastric cancer is very poor because of the lack of credible early diagnosis and therapy. Biological carcinogen (H. pylori) and chemical carcinogen (salt and nitrite) are the factors involved in gastric carcinogenic induction. In contrast, polyphenols (green tea) and vitamins are the factors involved in gastric carcinogenic prevention. Molecular diagnostics and prediction for tumor occurrence has become a hot area of cancer research in recent years. It is still unknown whether both gastric carcinogenic induced factors and gastric carcinogenic inhibitory factors could act on the same target molecule. Our research mainly focused on the effects of H. pylori infection, high salt stimulus or EGCG treatment on tumor proliferation related protein CIP2A expression level. Our aim is to discover that CIP2A could act as the same target molecule of gastric carcinogenic induced factors and gastric carcinogenic inhibitory factors. The following is the main research data and experiment results.1. The upregulation effects of H. pylori infection on CIP2A expressionH. pylori virulence factor CagA interacts with many signal molecules and elicits a series of cellular events. These events include the Ras/mitogen-activated protein (MAP) kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway activation,β-catenin activation, Src kinase activation, NF-κB pathway activation, and p38 MAP kinase pathway activation. H. pylori infection could affect the expression level of gastric carcinogenic factors, such as MYC,β-catenin and Cyclin D1. But up to now, the relationship between H. pylori infection and CIP2A expression has never been studied. In order to elucidate the relationship between these two factors, we designed H. pylori infection and CagA transfection in gastric cells and tried to investigate whether main virulence factor CagA of H. pylori could increase the expression of oncoprotein CIP2A in cell lines. Furthermore, we used signal inhibitors to examine which pathway was involved in the CagA-mediated regulation of CIP2A expression. Overall, it is the first time the mechanism by which H. pylori infection and bacterial oncoprotein CagA upregulated CIP2A expression in gastric cell lines has been elucidated. CagA protein activated the Src and MEK/ERK signal pathways resulting in the elevation of expression of CIP2A protein in AGS cells. This work has contributed to understanding the mechanism of gastric tumors caused by H. pylori infection in humans.2. H. pylori infection-induced-CIP2A upregulation is enhanced by high saltIt has been elucidated in chapter 1 that H. pylori infection and bacterial oncoprotein CagA could upregulate CIP2A expression in gastric cell lines. These results provide further evidence for the understanding of the molecular mechanism of H. pylori infection-induced-gastric carcinogenesis. As mentioned above, high salt intake is one of the risk factors involved in gastric carcinogenesis. Besides that, high salt stimuli and H. pylori infection could act as synergistic factors for gastric carcinogenesis. In order to elucidate the effects of high salt stimuli on H. pylori virulence factors, and observe the enhancement effects of high salt stimuli on H. pylori infection-induced-CIP2A upregulation, we designed some analysis, including the analysis of H. pylori virulence factors after treatment by different concentrations of salt in cultures and the analysis of CIP2A expression level in cells after infection by H. pylori strains which has been treated by different concentrations of salt in cultures. The results showed that high salt stimuli could increase the expression level of H. pylori virulence genes (cagA, vacA, tlpB, tip-alpha). More important is that H. pylori infection-induced-CIP2A upregulation is enhanced by high salt. The data about CIP2A analysis make further evidence for the understanding of synergistic effects of high salt stimuli and H. pylori infection in gastric carcinogenesis.3. EGCG inhibits tumor cell proliferation through its inhibitory effects on CIP2AIt has been reported that the molecular mechanism of EGCG-induced-tumor inhibition is related to many key proteins, such as beta-catenin, Myc, Cyclin Dl, matrix metalloproteinases (MMPs), P53, etc. As we all known, both BMX/Etk tyrosine kinases and newly identified oncoprotein CIP2A belong to the key factors which could regulate tumor cell proliferation. Up to now, the relationship between tumor inhibitor EGCG, tumor promoter BMX kinases, and oncoprotein CIP2A has never been discovered. Therefore, the studies about the effects of EGCG on BMX and CIP2A, or interaction between BMX and CIP2A could help us to understand the molecular mechanism of EGCG-induced-tumor cell proliferation inhibition. Based on this purpose, western blot analysis was used to verify the effects of EGCG on BMX and CIP2A; Eukaryotic expression plasmid and siRNA were employed to observe the interaction between BMX and CIP2A; Clonogenicity analysis was performed to investigate the role of CIP2A in the EGCG-induced-tumor cell proliferation inhibition. In view of our results, it is the first time that we proved the inhibitory effects of EGCG on CIP2A and BMX. And we also verified that over-expression of BMX could increase the expression of CIP2A. Moreover we investigated the key factors which could be involved in the molecular mechanism of EGCG-inhibited-tumor cells proliferation. The studies provide further evidence for the understanding of the molecular mechanism of EGCG-induced-tumor inhibition, and also provide strong basis for the guidance of EGCG usage in clinical therapy.更多还原