【作者】 于秀文；

【导师】 袁媛；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2009， 博士

【摘要】 前言幽门螺杆菌(Helicobacter pylori H.pylori)是1983年澳大利亚学者Warren和Marshall从慢性活动性胃炎病人胃粘膜活检标本中发现并分离,之后引起了全世界许多研究者的普遍关注。近年来研究表明,幽门螺杆菌感染是诱发胃癌重要的危险因素,1994年世界卫生组织下属的国际癌症研究机构(IARC)已将其列为人类胃癌的Ⅰ类致癌物。胃癌是常见的恶性肿瘤之一,其组织学类型多种多样,各具特征。Lauren’s分型是与胃癌的组织结构、细胞形态密切相关的一种简单有效的分型方法,将其分为肠型和弥漫型。肠型胃癌癌细胞一般排列成明显的腺管样结构,癌细胞呈柱状或立方形,结构类似肠癌。弥漫型胃癌癌细胞呈弥漫性生长,一般不形成腺管样,缺乏细胞连接,分化较差。那么肠型、弥漫型胃癌是如何发生的?发生发展过程如何呢?迄今尚未清楚。目前认为,肠型胃癌起源于肠化生粘膜,而弥漫型胃癌起源于胃固有粘膜。但也有人提出,胃癌来自腺颈部的干细胞。根据基因活化学说,此种细胞具有胃型和肠型上皮细胞两种基因,在干细胞癌变过程中如具有肠型上皮特性的基因被激活,则形成肠型胃癌。具有胃型上皮特性的基因被激活,即形成弥漫型胃癌。文献报道肠型胃癌和弥漫型胃癌的发生机制不同。肠型胃癌的癌变始于粘膜表层,经由胃炎→黏膜上皮和腺体萎缩→肠上皮化生→异性型增生→胃癌,而H.pylori寄生于粘膜表层,这可能是肠型胃癌与H.pylori感染关系密切的原因之一。弥漫型胃癌则不经历上述过程,为再生的胃黏膜上皮异型增生,病变起始于粘膜深层,是由于遗传因素所决定的。那么H.pylori感染对胃癌组织学发生有何影响,究竟导致何种类型胃癌,其机制如何呢?一些学者认为,H.pylori感染与肠型胃癌发生有关。还有一些学者认为,H.pylori感染与肠型、弥漫型胃癌均有关,或与胃癌Lauren’s分型无关。目前研究发现,H.pylori感染后可以通过不同的信号转导途径引起胃癌的发生。Wnt/β-catenin信号转导途径是对细胞的分化、迁移、增殖、极性起着重要调节作用的途径。是由最上游的Wnt蛋白启动激活,通过β-catenin在细胞质内聚集,转位进入细胞核,与TCF/LEF结合,促使下游靶基因增高,从而引起细胞增殖。Saitoh T等研究指出,H.pylori感染胃粘膜时,引起IFNγ、TNFα的含量增加,TNFα上调Wnt10B的表达,然后通过β-catenin/TCF信号途径引起胃癌的发生。还有研究表明,APC/β-catenin/TCF在胃癌发生过程中尤其在肠型胃癌发生中起到重要作用。那么,H.pylori感染是否可以通过Wnt/β-catenin途径和胃癌的组织学分型相关呢?目前尚不清楚。目的探讨H.pylori感染对胃癌组织学发生的影响以及H.pylori感染引起不同类型胃癌组织学发生的机制,为胃癌的预防和治疗提供理论依据。方法本研究选用辽宁省庄河地区临床医院经胃镜取材的胃粘膜标本,利用HE及免疫组织化学染色方法检测H.pylori,利用免疫组织化学染色方法同步检测E-cadherin、β-catenin、TCF4蛋白表达。采用细胞培养、细菌培养及细菌与细胞共培养制作H.pylori感染体外细胞转化模型,利用相差显微镜、普通显微镜及电子显微镜观察细胞形态学变化;利用MTT、Ki67免疫组化、平板克隆实验检测细胞增殖转化情况;利用免疫荧光双标染色观察H.pylori感染后GES-1细胞的E-cadherin、β-catenin、TCF4蛋白表达情况;利用RT-PCR方法检测H.pylori感染后GES-1细胞E-cadherin、β-catenin、TCF4在mRNA水平表达情况。统计学分析采用χ~2检验、Fisher’s Exact Test比较计数资料的组间差异,Mann-Whitney Test检测计量资料组间差异,各个因子的相关性采用Spearman相关分析。结果1、H.pylori感染率肠型胃癌高于弥漫型胃癌(χ~2=6.784,P=0.009)。2、E-cadherin,β-catenin,TCF4表达率均为肠型胃癌高于弥漫型胃癌,β-catenin在细胞膜、细胞质、细胞核的表达率均为肠型胃癌高于弥漫型胃癌,(P＜0.01)。3、肠型胃癌β-catenin细胞膜与细胞质(r=0.747,P=0.000)、细胞质与细胞核呈正相关(r=0.347,P=0.000);而E-cadherin与β-catenin细胞膜(r=0.012,P=0.906)、β-catenin细胞核与TCF4蛋白表达无相关性(r=0.146,P=0.143)。弥漫型胃癌E-cadherin与β-catenin细胞膜(r=0.225,P=0.025)、β-catenin细胞膜与细胞质(r=0.697,P=0.000)、细胞质与细胞核呈正相关(r=0.306,P=0.002);而β-catenin细胞核与TCF4蛋白表达无相关性(r=0.118,P=0.241)。4、肠型胃癌组织β-catenin细胞质蛋白、TCF4蛋白表达率H.pylori阳性组高于H.pylori阴性组(P＜0.05)。弥漫型胃癌E-cadherin蛋白表达率为H.pylori阳性组高于H.pylori阴性组(P＜0.05),β-catenin细胞质蛋白表达率为H.pylori阳性组低于H.pylori阴性组(P＜0.05)。5、H.pylori与GES-1共培养45天后出现形态学变化,表现为细胞大小不等,形态不规则,体积增大,偶见巨细胞;核分裂像增加,偶见病理性核分裂像;透射电子显微镜下,细胞微绒毛增多;细胞核增大,形态不规则,染色质增多,位于核膜下,核膜增厚。6、H.pylori与GES-1共培养45天后GES-1细胞增殖能力增强,表现为MTTOD值、Ki67表达的平均光密度值、平板克隆实验均为H.pylori作用后GES-1细胞高于GES-1对照细胞(P＜0.05)。7、免疫荧光结果显示,H.pylori作用后GES-1细胞E-cadherin、β-catenin、TCF4表达明显高于GES-1对照细胞:β-catenin表达部应出现变化。8、RT-PCR结果显示,H.pylori作用后GES-1细胞β-catenin、TCF4mRNA高于GES-1对照细胞;E-cadherin低于GES-1对照细胞。结论1、H.pylori感染率肠型胃癌高于弥漫型胃癌。2、肠型胃癌E-cadherin、β-catenin、TCF4蛋白表达率高于弥漫型胃癌,肠型胃癌H.pylori感染增加β-catenin、TCF4表达,弥漫型胃癌H.pylori感染增加E-cadherin表达及降低了β-catenin细胞质表达。3、H.pylori可以诱导GES-1细胞转化,并且出现肠型胃癌分化特征。4、H.pylori诱导GES-1细胞的转化与β-catenin/TCF4信号转导通路有关。更多还原

【Abstract】 BackgroundHelicobacter pylori(H.pylori) is separated and then identified from gastric mucous specimens of chronic gastritis patients by the Australian scholars Warren and Marshall in 1983,and is concerned by much researchers all over the world.In recent years,some studies have shown that Helicobacter pylori infection is an important risk factor induced gastric cancer(GC),and it have been classified as carcinogen category I for human gastric cancer in 1994 by the World Health Organization’s affiliated International Agency for Research on Cancer(IARC).Gastric cancer is a common malignant tumor,and its histologic types varied from each other for everyone with its own characteristics.Lauren’s classification is a simple and effective method of classification which is related to the organizational structure and cell morphology of gastric cancer,it divides GC into intestinal type and diffuse type.The cells of intestinal type GC generally arrange in a clear tubular-like structure,they are columnar or cubic structure which is similar to intestinal cancer.The cells of diffuse-type GC grow diffusely,generally do not form a tubular-like structure,is lack of cell junction and poorly differentiate.Then how does intestinal type and diffuse type GC happen? How about the incident and development process? It was not clear so far.At present,some scholars considere that intestinal type GC originates the mucosa of gastric intestinal metaplasia,and diffuse-type GC originates the gastric inherent mucosa.However,other scholars suggest that GC come from stem cells in the neck of gastric glands.Such stem cells possess two kinds of genes which were from the stomach and intestinal epithelial cells respectively according to the theory of gene activation.In the process of stem cells cancerization,if the gene possesses intestinal epithelial characteristics is activated,it would form intestinal type GC;if the gene possesses gastric-type epithelial characteristics is activated,it would form diffuse-type GC. What does H.pylori infection impact on the occurrence of gastric cancer,what types of GC does H.pylori lead to,and what mechanism would it be? Most scholars think H.pylori infection primarily be related to the occurrence of intestinal type GC. Some other scholars also think it is related to both intestinal type and diffuse-type of GC,or think it is not related to Lauren’s classification of GC.The occurrent mechanism of intestinal type GC reported in the literature is different from diffuse-type of GC.The cancerization of intestinal-type GC begins from the mucosal surface,the process is from gastritis-mucosal epithelium and glandular atrophy-intestinal metaplasia -atypical hyperplasia-gastric cancer,and as H.pylori parasitising on the mucosal surface,it may be one of the reasons for intestinal type GC is closely related to H.pylori infection.The diffuse-type of GC is not going through the process above,it comes from the regeneration of gastric epithelial dysplasia,and begins in the deep mucosa,this is due to genetic factors.Then why GC caused by H.pylori infection is not all intestinal type, but also diffuse-type GC? This may be related to the different genes carried by different individuals as well as the changes of different genes caused by H.pylori infection.What mechanisms does H.pylori infection cause the different histological occurrence of gastric cancer? The current study shows that,H.pylori infection can caused the occurrence of GC through different signal transduction pathways.The signal transduction pathway Wnt/β-catenin plays an important role in the regulation of cell differentiation,migration,proliferation and polarity.This pathway is activated by the Wnt protein upstream,throughβ-catenin accumulation in the cytoplasm,translocation into the nucleus,combination with TCF/LEF,increasing downstream target genes,thus leading to cell proliferation.Saitoh T and his collegues found that when H.pylori infected gastric mucosa,IFNγ,TNFαlevels were caused increasing,and TNFαincreased the expression of Wnt10B,and then caused the occurrence of gastric cancer throughβ-catenin/TCF signaling pathways.The current study showed that, APC/β-catenin/TCF played an important role in the occurrence of gastric cancer, especially in intestinal type GC.Whether H.pylori infection can be related to the occurrence of histological type of GC through Wnt/β-catenin pathways? It was not clear.AimsThe purpose of this study is the effect of H.pylori infection to histological occurrence of gastric cancer,the mechanisms of different histological types of gastric cancer caused by H.pylori infection,providing a theoretical basis on the prevention and treatment of gastric cancer.MethodsThis study selected the gastric mucosa specimens derived by endoscopy from the Hospitals in Zhuanghe Area in Liaoning Province,the detection of H.pylori infection using HE and immunohistochemical method,the detection of E-cadherin,β-catenin, TCF4 protein expression using immunohistochemical staining.The transformation model of H.pylori infection in vitro used the techniques of cell culture,bacterial culture and bacterial and cell co-culture,the observation of cell morphological changes using phase-contrast microscopy and ordinary microscope and electron microscope;the detecion of proliferation and transformation situation using MTT,Ki67 immunohistochemistry,flat cell cloning assay;the E-cadherin,β-catenin, TCF4 protein expression in GES-1 cells infected by H.pylori using double-labeled immunofluorescence staining;the E -cadherin,β-catenin,TCF4 expression at mRNA level in GES-1 cells infected by H.pylori using RT-PCR method.The statistical analysis usedχ~2 test,Fisher’s Exact Test for the comparison of the count data between groups,Mann-Whitney Test for the comparison of the measurement data between groups,Spearman correlation analysis for the relevance of each factors.Results1.H.pylori infection rate in the patients with intestinal type gastric cancer was higher than that of the diffuse-type GC(χ~2=6.784,P=0.009).2.The expression rates of E-cadherin,β-catenin,TCF4 protein were higher in the intestinal GC than that of the diffuse-type one.the cell membrane,cytoplasm and nucleus ofβ-catenin were higher in the intestinal GC than that of the diffuse-type(P <0.01).3.In intestinal type GC,there was positively correlation between in the cell membrane and in the cytoplasm ofβ-catenin(r=0.747,P=0.000),between in the cytoplasm and in the nucleus(r=0.347,P=0.000);and there was no correlation between E-cadherin protein and in the cell membrane ofβ-catenin(r=0.012,P=0.906),betweenβ-catenin protein and TCF4 in the nucleus(r=0.146,P=0.143).In diffuse-type GC there was positively correlation between E-cadherin protein and in the cell membrane ofβ-catenin(r=0.225,P=0.025),between in the cell membrane and in the cytoplasm ofβ-catenin(r=0.697,P=0.000),between in the cytoplasm and the nucleus ofβ-catenin(r=0.306,P=0.002);and there was no correlation between in the nuclear ofβ-catenin and TCF4 protein expression(r=0.118,P=0.241).4.In intestinal type GC,β-catenin protein in the cell cytoplasm and TCF4 protein expression rates in H.pylori-positive group were higher than the rates in H.pylori negative group(P<0.05).In diffuse-type GC,E-cadherin protein expression rate in H.pylori-positive group was higher than that of in the H.pylori-negative group(P<0.05),β-catenin protein expression rate in the cytoplasm H.pylori positive group was lower than the H.pylori- negative group(P<0.05).5.After co-culturing H.pylori and GES-1 cell 45 days,there emerged morphological changes,reflecting as the cells different in size,the cell shape irregular, and the cell size increasing,occasionally appearring giant cells;karyokinesis increasing and occasionally appearring pathological karyokinesis.In the transmission electron microscope,the cells microvilli increasing;the cell nucleus increasing,the cell shape irregular,the chromatin increasing which was located under the nuclear membrane,and the nuclear membrane thickening.6.After co-culturing H.pylori and GES-1 cell 45 days,the GES-1 cells MTT OD value,the cells expressed Ki67 and in the plate cloning experiment,GES-1 cells affected by H.pylori higher than that of the GES-1 control cells.7.The results by immunofluorescence showed that,E-cadherin,β-catenin,TCF4 expression in GES-1 control cells was significantly lower than GES-1 cells affected by H.pylori;theβ-catenin expression location changed.8.The results by by RT-PCR showed that,β-catenin,TCF4 expression in GES-1 control cells was significantly lower than GES-1 cells affected by H.pylori;E-cadherin higher than GES-1 cells affected by H.pylori.Conclusion1.H.pylori infection rate in intestinal type GC is higher than the diffuse-type GC.2.The expression rates of E-cadherin,β-catenin and TCF4 protein are higher in the intestinal GC than that of the diffuse-type one.In intestinal type GC H.pylori infection increasesβ-catenin and TCF4 expression,in diffuse-type GC H.pylori infection increases E-cadherin expression and reduces the cytoplasmic expression ofβ-catenin.3.H.pylori can induce the transformation of GES-1 cells,and appear the characteristics of intestinal type GC.4.The transformation of GES-1 cells induced by H.pylori may accomplish throughβ-catenin/TCF4 signal pathway.更多还原