【作者】 薛丽英；

【导师】 张祥宏；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 胃癌是最常见的恶性肿瘤之一。近几十年来,胃癌总体发生率有所下降,但胃癌的发生部位出现了明显的变化,主要表现在来源于胃远端部位（胃窦）胃癌的发生率明显下降,而食管胃交界部位（esophagogastric junction, EGJ）远端食管腺癌和贲门腺癌的发生率呈明显增高趋势,此变化趋势在西方国家尤为明显。初步研究结果表明,我国居民胃癌的发生部位也出现类似的变化,从上世纪90年代后我国北方太行山区居民贲门腺癌（gastric cardiac adenocarcinoma, GCA）的发病率呈现逐渐增高的趋势,具体机制尚不清楚。最近有研究提示,贲门腺癌在肿瘤的发生机制和生物学行为方面可能不同于非贲门部位的胃癌,为一种独特的胃癌病理学亚型。Maeda等发现贲门腺癌淋巴结转移率比远端胃腺癌（gastric distal adenocarcinoma, GDA）高,且预后不良。从病因学角度Hp感染是远端胃腺癌发生的重要危险因素,而在贲门腺癌发生中的作用却存在很大争议。因此,探讨不同部位胃癌在生物学行为、免疫表型和一些关键分子作用机制方面的可能差异,对进一步揭示胃癌发生部位变化的可能机制以及胃癌高发区居民胃癌的合理防治具有非常重要的意义,是肿瘤工作者面临的一个重要挑战。细胞周期调控异常是恶性肿瘤形成的一个重要机制。P16^INK4a和p14^ARF为INK4a/ARF家族成员,二者作为细胞周期调控蛋白,作用于细胞中两条最主要细胞周期调控通路Rb通路(p16^INK4a-Rb)和p53(p14^ARF-p53)通路,在肿瘤的发生、发展过程中起着关键作用。p16^INK4a和p14^ARF表达失活可见于多种人类肿瘤中,比如:皮肤癌、胰腺癌、乳腺癌、食管癌、肺癌、淋巴瘤、结直肠癌等。胃肠道肿瘤中p16^INK4a和p14^ARF表达与肿瘤组织的分化程度、组织分型等密切相关。目前尚未见有关p16^INK4a和p14^ARF在贲门腺癌和远端胃腺癌中表达的对比研究。细胞角蛋白（Cytokeratins,CKs）和黏蛋白（Mucins,MUC）是确定胃肠道癌免疫表型特征常用的标志物。角蛋白是上皮细胞中的骨架蛋白之一,是上皮细胞的特征性标志物,由分子量不同的角蛋白组成一个庞大的家族,依据上皮细胞类型的不同角蛋白谱不同,可依此确定肿瘤的病理组织学类型和分化方向。黏蛋白是分布于上皮细胞表面高度糖化、高分子量的糖蛋白,有MUC1、MUC2等多种类型,其中MUC2为分泌型黏蛋白,主要表达于肠杯状细胞,为胃黏膜肠上皮化生表面标志物,与胃癌组织学类型密切相关。为探讨贲门腺癌和远端胃腺癌在发生发展中存在的可能差异,本研究中我们从四个方面对不同部位胃癌组织进行了对比研究。主要包括:1贲门腺癌与远端胃腺癌组织CKs和MUCs免疫表型的对比分析;2 p14^ARF和p16^INK4a蛋白在贲门腺癌和远端胃腺癌组织中的表达及意义;3 p14^ARF和p16^INK4a基因在上述不同部位胃癌组织中缺失情况的对比分析;4贲门腺癌与远端胃腺癌组织p14^ARF和p16^INK4a基因甲基化的对比研究。本文拟通过探讨不同部位胃癌之间存在的差异,揭示我国贲门腺癌发病率升高的原因,为贲门腺癌的防治提供理论依据。本研究论文分为四个部分:第一部分:贲门腺癌与远端胃腺癌CKs和MUCs免疫表型的对比分析目的:通过各种不同CKs和MUCs表达变化的对比分析,探讨贲门腺癌和远端胃腺癌在免疫表型方面的差异。方法:选取2006-2009年河北省磁县县医院、赞皇县医院以及河北医科大学第二医院手术切除胃癌组织标本共99例,其中贲门腺癌50例（肿瘤中心在Z线上1cm和下2cm以内）,远端胃腺癌49例。运用免疫组织化学方法研究各种不同CKs分子（CK7、CK14、CK19和CK20）和MUCs（MUC1、MUC2和MUC5AC）在贲门腺癌与远端胃腺癌中的表达情况,对比分析不同部位胃癌组织之间CKs、MUCs免疫表型差异性。结果:1.1 CKs和MUCs的表达在贲门腺癌与远端胃腺癌组织中的对比分析免疫组织化学检测结果表明,CK14、MUC2在贲门腺癌与远端胃腺癌中的阳性表达率明显不同。CK14在贲门腺癌组织中的阳性表达率明显高于远端胃腺癌组织（30.0% vs 10.2%, P<0.05）,而MUC2的阳性表达率在远端胃腺癌组织中显著高于贲门腺癌组织（71.4% vs 52.0%, P<0.05）。进一步分析发现,CK14的表达在贲门腺癌组织中与肿瘤组织分化程度以及有无淋巴结转移相关。中低分化贲门腺癌肿瘤组织CK14阳性表达率明显低于高分化肿瘤组织（22.0% vs 66.7%, P<0.05）;伴有淋巴结转移的组织CK14阳性表达率比无淋巴结转移者明显降低（17.9% vs 45.5%, P<0.05）。而在远端胃癌组织中CK14表达与组织分化程度和淋巴结转移均无相关性（P>0.05）。除CK14和MUC2外,其他相关分子包括:CK7、CK19、CK20、MUC1和MUC5AC的表达在贲门腺癌与远端胃腺癌中均未见显著差异（P>0.05）。1.2贲门腺癌和远端胃腺癌中CK7、CK20以及CK7/CK20联合表达特征的对比研究分别对CK7和CK20在不同部位胃癌中的表达进行比较,结果表明CK7和CK20在贲门腺癌组织以及远端胃腺癌组织中阳性表达率均无明显差异（CK7:86.0% vs 79.6%, P>0.05;CK20:56.0% vs 40.8%, P>0.05）。在远端胃腺癌组织中CK20的表达与组织分化程度相关。中低分化程度癌组织CK20阳性表达明显低于高分化腺癌（33.3% vs 70.0%, P<0.05）。而在贲门腺癌组织中,CK20表达与组织分化程度及有无淋巴结转移无明显相关（P>0.05）。CK7/CK20联合表达特征的分析表明,50例贲门腺癌中40.0%为CK7+/CK20-,46.0%为CK7+/CK20+,10.0%为CK7-/CK20+,仅4.0%为CK7-/CK20-。远端胃腺癌中四种免疫表型表达分别为46.9%、32.7%、8.2%和12.2%。CK7/CK20联合表达在贲门腺癌和远端胃腺癌中无明显差异（P>0.05）,而且与不同部位胃癌分化程度无明显相关性（P>0.05）。第二部分:贲门腺癌与远端胃腺癌组织p14^ARF和p16^INK4a蛋白表达及其意义的对比研究目的:比较贲门腺癌和远端胃腺癌组织中p16^INK4a和p14^ARF蛋白表达的差异。方法:重新选取贲门腺癌44例和远端胃腺癌40例,同时取正常胃黏膜（同期胃癌手术切除的未受累的胃残端）25例作为对照。采用免疫组织化学方法检测p16^INK4a和p14^ARF蛋白在不同部位胃癌中的表达情况。结果:2.1贲门腺癌与远端胃腺癌中p14^ARF蛋白表达的比较2.1.1 p14^ARF在贲门腺癌与远端胃腺癌组织中的表达P14^ARF蛋白阳性反应定位于细胞核和/或细胞浆。正常对照胃黏膜组织中p14^ARF阳性表达率明显高于胃癌组织（84.0% vs 45.2%, P<0.05）。在不同部位的胃癌病例中,贲门腺癌中阳性表达率明显低于远端胃腺癌（34.1% vs 57.5%, P<0.05）。2.1.2贲门腺癌和远端胃腺癌p14^ARF表达的临床病理意义的对比分析:远端胃腺癌中p14^ARF蛋白的表达与患者年龄、性别、肿瘤组织分化程度及淋巴结转移均无明显相关（P>0.05）。而贲门腺癌组织中,低分化腺癌p14^ARF阳性表达率明显低于高分化腺癌（18.5% vs 58.8%, P<0.05）;伴有淋巴结转移癌组织阳性表达比无淋巴结转移明显降低（17.4% vs 52.4%, P<0.05）。贲门腺癌中p14^ARF蛋白的表达与患者年龄、性别无明显相关（P>0.05）。p14^ARF阳性表达率在低分化的贲门腺癌和低分化远端胃腺癌组织的表达存在差异,低分化远端胃腺癌组织比贲门腺癌组织p14^ARF蛋白表达明显增高（55.2% vs 18.5%, P<0.05）。同时,伴有淋巴结转移的贲门腺癌组织p14^ARF阳性表达明显低于远端胃腺癌（17.4% vs 57.1%, P<0.05）。2.2贲门腺癌和远端胃腺癌中p16^INK4a蛋白表达的比较2.2.1 p16^INK4a在贲门腺癌与远端胃腺癌组织中的表达情况免疫组织化学结果显示,胃癌组织中p16^INK4a蛋白阳性表达率为58.3%（49/84）,比正常胃黏膜表达明显降低（96.0%, P<0.05）。贲门腺癌组织p16^INK4a阳性表达明显低于远端胃腺癌（43.2% vs 75.0%, P<0.05）。2.2.2 P16^INK4a表达与贲门腺癌和远端胃腺癌临床病理资料关系的对比分析p16^INK4a蛋白在贲门腺癌和远端胃腺癌中的表达与患者年龄、性别、肿瘤组织分化程度及淋巴结转移均无明显相关（P>0.05）。低分化贲门腺癌中p16^INK4a表达比低分化远端胃腺癌中明显降低（37.0% vs 72.4%, P<0.05）。伴有淋巴结转移的贲门腺癌中p16^INK4a阳性表达明显低于伴有淋巴结转移的远端胃腺癌（39.1% vs 75.0%, P<0.05）。第三部分:贲门腺癌与远端胃腺癌组织p14^ARF和p16^INK4a基因缺失的比较目的:比较贲门腺癌和远端胃腺癌中p16^INK4a和p14^ARF抑癌基因位点缺失。方法:选取贲门腺癌44例和远端胃腺癌40例,取正常胃黏膜（同期胃癌手术切除的未受累的胃残端）15例作为对照。分别提取组织DNA,采用PCR方法检测p16^INK4a（E1α和E2外显子）和p14^ARF外显子（E1β和E2外显子）的缺失情况。结果:3.1贲门腺癌和远端胃腺癌组织中p16^INK4a基因E1α和E2外显子缺失的对比PCR结果显示,正常胃黏膜组织E1α和E2未见缺失。84例胃癌中E1α、E2单纯性缺失率分别为9.5%（8/84）和8.3%（7/84）。p16^INK4a基因E1α和E2外显子总缺失率为17.9%（15/84）。在44例贲门腺癌中,E1α缺失4例（9.1%）,E2缺失5例（11.4%）,E1α和E2总缺失率为20.5%（9/44）。而在40例远端胃腺癌组织中E1α、E2单纯性缺失分别为10.0%（4/40）和5.0%（2/40）,E1α和或E2总缺失率为15.0%（6/40）。统计结果显示,在贲门腺癌和远端胃腺癌中E1α、E2单纯缺失及p16^INK4a基因E1α和E2外显子总缺失率均无明显差异（P>0.05）。3.2贲门腺癌和远端胃腺癌组织中p14^ARF基因E1β和E2外显子缺失的对比正常胃黏膜组织中E1β、E2未见缺失。84例胃癌中E1β、E2单纯性缺失率分别为6.0%（5/84）和8.3%（7/84）。p14^ARF基因E1β和E2外显子缺失率为14.3%（12/84）。在贲门腺癌中,E1β缺失3例（6.8%）,第二外显子E2缺失5例（11.4%）。p14^ARF基因E1β和E2外显子总的缺失率为18.2%（8/44）;而在远端胃腺癌组织中E1β、E2单纯性缺失率分别为5.0%（2/40）和5.0%（2/40）,p14^ARF基因E1β和E2外显子总缺失率为10.0%（4/40）。统计结果显示,贲门腺癌和远端胃腺癌中E1β、E2单纯性缺失及p14^ARF基因E1β和E2外显子总缺失率无明显差异（P>0.05）。3.3 p14^ARF和p16^INK4a在胃癌中的表达与基因缺失的关系分析在84例胃癌中,p16^INK4a蛋白表达阴性者为35例,p16^INK4a基因E1α和E2外显子总缺失为15例,并且在这15例胃癌组织中p16^INK4a蛋白的表达均为阴性。在84例胃癌中,p14^ARF蛋白表达阴性者为46例,而p14^ARF基因E1β和E2外显子总缺失为12例,12例胃癌组织中p14^ARF蛋白的表达均为阴性。上述结果提示,p14^ARF和p16^INK4a基因缺失是引起其蛋白表达失活的机制之一。第四部分:贲门腺癌与远端胃腺癌组织p16^INK4a和p14^ARF基因甲基化的对比研究目的:比较不同部位胃癌中p16^INK4a和p14^ARF基因CpG岛甲基化情况,分析贲门腺癌和远端胃腺癌中p16^INK4a和p14^ARF失活的机制。方法:本实验采用甲基化特异性PCR（Methylmion Specific PCR,MSP）对52例贲门腺癌、39例远端胃腺癌以及15例正常胃黏膜组织中p16^INK4a和p14^ARF甲基化水平进行检测;同时提取10例贲门腺癌和8例远端胃腺癌新鲜组织总RNA,运用Real-time PCR方法检测胃癌组织中p16^INK4a和p14^ARF mRNA的表达。结果:4.1 p14^ARF启动子区甲基化检测结果4.1.1 p14^ARF在贲门腺癌与远端胃腺癌组织中甲基化水平的比较MSP检测结果发现,15例正常胃黏膜中有2例发生p14启动子区甲基化,甲基化率为13.3%;胃癌组织p14^ARF甲基化率为56.0%（51/91）,明显高于正常胃黏膜（P<0.05）。贲门腺癌和远端胃腺癌的甲基化率分别为61.5%和43.6%,二者均比正常胃黏膜p14^ARF甲基化率增高（P<0.05）。同时,贲门腺癌组织中p14^ARF甲基化水平高于远端胃腺癌（P<0.05）。4.1.2 p14^ARF甲基化与贲门腺癌和远端胃腺癌临床资料的关系远端胃腺癌中p14^ARF甲基化率与患者年龄、性别、肿瘤组织分化程度及淋巴结转移均无明显相关（P>0.05）。贲门腺癌组织中,低分化腺癌p14^ARF甲基化率为78.1%,明显高于高分化腺癌（45.5%, P<0.05）。贲门腺癌中p14^ARF甲基化率与患者年龄、性别及淋巴结转移均无明显相关（P>0.05）。进一步研究发现,低分化贲门腺癌与低分化远端胃腺癌组织相比,p14^ARF甲基化率明显不同。在低分化贲门腺癌组织中p14^ARF甲基化率明显高于低分化远端胃腺癌组织（78.1% vs 48.0%, P<0.05）。4.2胃癌中p16^INK4a启动子区甲基化检测结果4.2.1 p16^INK4a在贲门腺癌与远端胃腺癌组织中的甲基化情况结果表明,91例胃癌组织中有58例发生p16^INK4a甲基化现象,甲基化率为63.7%,比正常胃黏膜p16^INK4a甲基化明显增高（20.0%, P<0.05）。贲门腺癌和远端胃腺癌组织中p16^INK4a甲基化阳性率分别为73.1%和51.3%,均高于正常胃黏膜组织（P<0.05）。同时,贲门腺癌组织中p16^INK4a甲基化率明显高于远端胃腺癌,统计有显著差异（P<0.05）。4.2.2 p16^INK4a甲基化与贲门腺癌和远端胃腺癌临床资料的关系贲门腺癌组织和远端胃腺癌组织中,p16^INK4a甲基化与患者年龄、性别、肿瘤组织分化程度及淋巴结转移均无明显相关（P>0.05）。进一步研究发现,低分化程度贲门腺癌组织p16^INK4a甲基化明显高于远端胃腺癌（81.3% vs 56.0%;P<0.05）。4.3胃癌中p14^ARF和p16^INK4a基因甲基化与其mRNA表达的相关性分析选取18例新鲜组织,其中贲门腺癌10例、远端胃腺癌8例。根据MSP检测结果显示p14^ARF甲基化阳性10例,阴性8例;p16^INK4a甲基化阳性9例,阴性9例。采用Real-time PCR技术检测了p14^ARF和p16^INK4a mRNA的表达情况,发现发生甲基化的胃癌组织p14^ARF和p16^INK4a mRNA水平均明显低于非甲基化组。结果提示胃癌组织中p14^ARF和p16^INK4a启动子区甲基化可致其mRNA表达明显降低（P<0.05）。结论:1贲门腺癌组织中CK14表达明显高于远端胃腺癌,而MUC2表达明显低于远端胃腺癌组织,提示贲门腺癌可能在组织分型及组织学来源方面与远端胃腺癌有所不同。2贲门腺癌组织中p14^ARF和p16^INK4a蛋白的表达均明显低于远端胃腺癌组织,且p14^ARF的表达与肿瘤分化程度及淋巴结转移密切相关;而在远端胃腺癌中其表达与肿瘤分化程度及淋巴结转移均无明显相关。因此,贲门腺癌与远端胃腺癌组织中抑癌基因p14^ARF和p16^INK4a在蛋白水平上的表达及其意义均存在差异。3 p14^ARF和p16^INK4a基因外显子缺失的贲门腺癌和远端胃腺癌组织中,其相应蛋白的表达也为阴性。但是,在贲门腺癌和远端胃腺癌中p14^ARF和p16^INK4a基因缺失率无明显差异,可能与胃癌发生部位无关。4 p14^ARF和p16^INK4a基因甲基化水平在贲门腺癌组织中明显高于远端胃腺癌组织,提示启动子区甲基化水平的差异可能是贲门腺癌与远端胃腺癌p14^ARF和p16^INK4a表达差异的原因之一。更多还原

【Abstract】 During the past decades, the incidence of gastric cancer has been decreased with the change of the life-style. But the subsites of gastric carcinoma underwent significant changes. The incidence of adenocarcinoma at antrum or distal stomach has been decreased, while that at esophagogastric junction （EGJ, the proximal stomach and distal esophagus） was increased in most developed countries. In 1996, Siewert definited the adenocarcinomas located in EGJ as adenocarcinoma of esophagogastric junction （AEG） and classified them into three types （Type I, II and III） according to the tumor center location. In the past several years, the incidence of adenocarcinoma of EGJ region was also found to be increased in the high incidence area of esohageal and gastric cancer in China, but the reason was still unclear. In our country, the absolute majority of the adenocarcinomas of esophagogastric junction belong to Siewert II and III subtype, so we generally refered the adenocarcinomas in these subsites as gastric cardiac adenocarcinoma （GCA）.Recent study showed that GCA was different from other gastric cancers in many ways, such as biological behavior and etiological factors. Accordingly, it might be a special subtype or independent entity of gastric carcinoma. Maeda et al reported that GCA has a higher rate of lymph node metastasis and unfavourable prognosis than noncardia tumors. Meanwhile, Hp infection was proven to be a key factor in gastric distal adenocarcinomas （GDA）, while the role of it involed in GCA is controversial now. Up to now, little study about the incidence and putative differences between GCA and GDA was conducted in China.As we all know, abnormality of cell cycle is an important mechanism in carcinogenesis. P16^INK4a and p14^ARF, the members of INK4a/ARF family, could regulate the cell cycle distribution by participating in the pathway of Rb (p16^INK4a-Rb) and p53 (p14^ARF-p53), and play the critical role in tumorigeness and progression. Inactivation of p16^INK4a and p14^ARF could be detected in many human tumors including skin cancer, pancreatic cancer, pancreatic cancer, esophageal carcinoma, lung cancer. Moreover, inactivation of p16^INK4a and p14^ARF was also found in gastroenteric tumor, and the expression of p16^INK4a and p14^ARF was associated with histological differentiation and histotype in gastric carcinomas. By now, there is no study on the expression of p16^INK4a and p14^ARF between GCA and GDA. In addition, Cytokeratins （CK） is used as diagnostic marker in distinguishing different types of carcinomas, as its composition is highly heterogeneous depending on epithelial cell type. Mucins （MUC） are high molecular weight glycoproteins at surface epithelia that play important roles in carcinogenesis, Especially, MUC2 is a marker of intestinal metaplasia regarding of its staining in intestinal goblet cells, was related with histotype in gastric carcinomas.So in this study, we studied the possible difference between GCA and GDA in tumorigeness and progression in human primary gastric carcinomas.The contents of this study included the following four parts: 1. Comparison of the expression of immunophenotypic markers including CKs and MUCs between GCA and GDA. 2. Differences in the expression of p14^ARF and p16^INK4a between GCA and GDA. 3. The similarity and differences in the deletions of p16^INK4a and p14^ARF genes between GCA and GDA. 4. Difference of p14^ARF and p16^INK4a mythelations between GCA and GDA. The aim of this study is to explore the possible differences in the afore-mentioned four aspects between gastric cardiac and distal adenocarcinomas, to reveal the possible reason of the increased incidence of GCA in our country.This study includes four parts:PartⅠComparison of the expressions of immunophenotypic markers of CKs and MUCs between GCA and GDAObjective: To explore the possible difference of immunophenotype between GCA and GDA by detecting the expression of CKs and MUCs. Methods: Ninety-nine patients with gastric cardiac and distal adenocarcinomas consecutively operated at the second hospital of Hebei Medical Universty and Cixian, Zanhuang County Hospital in Hebei Province between January 2006 and December 2009 were included in the study. All patients were underwent a preoperative upper gastrointestinal endoscopy （with biopsy specimens routinely performed）. Fifty patients with cardiac carcinoma attended in this study were chosen according to the tumor center within 1 cm above and 2 cm below the anatomic esophagogastric junction （EGJ）. Forty-nine cases with the tumor at antrum were operated with different surgical approaches. To compare the different immunophenotype between GCA and GDA, the expression of CKs （CK7, CK14, CK19 and CK20） and MUCs （MUC1, MUC2 and MUC5AC） was detected by immunohistochemistry method.Results:1.1 The expression of CKs and MUCs in GCA in comparison with GDAImmunohistochemistry results showed that the expression of CK14 and MUC2 displayed significantly difference between GCA and GDA. The expression of CK14 was significantly higher in GCA than that in GDA （30.0% vs 10.2%, P<0.05）. Inversely, MUC2 expression was lower in GCA than that in GDA （52.0% vs 71.4%, P<0.05）. Furthermore, CK14 expression was associated with histological differentiation and lymph node metastasis in GCA. Expression of CK14 was significantly lower in GCA with moderate/poor differentiation than that in well differentiation （22.0% vs 66.7%, P<0.05）, and it was also decreased in GCA with lymph node metastasis compared to that without lymph node metastasis （17.9% vs 45.5%, P<0.05）. But CK14 expression was not related with the extent of differentiation in GDA （P>0.05）. As for the other molecules including CK7, CK19, CK20, MUC1 and MUC5AC, no significant differences could be found in their immunoreactivity between GCA and GDA （P>0.05）.1.2 Comparative Studies of CK7, CK20 and combined expression of CK7/CK20 between GCA and GDA The expression of CK7 and CK20 showed no significant differences between GCA and GDA （CK7: 86.0% vs 79.6%, P>0.05; CK20: 56.0% vs 40.8%, P>0.05）. In GDA, CK20 expression was associated with the differentiation of tumors. The expression of CK20 was lower in GDA with moderate/poor differentiation than that in well differentiation （33.3% vs 70.0%, P<0.05）. However, expression of CK20 was not related with the differentiation and lymph node metastasis in GCA （P>0.05）.The coexpression of CK7/CK20 results showed that the expression rate of CK7+/CK20-, CK7+/CK20+, CK7-/CK20+ and CK7-/CK20- in GCA was 40.0%, 46.0%, 10.0% and 4.0%, respectively. And the corresponding value of CK7/CK20 expression in GDA was 46.9%, 32.7%, 8.2% and 12.2%, respectively. These data showed no statistically significant difference within the two groups （P>0.05） and no distinct association with histological differentiation （P>0.05）.PartⅡThe difference of p16^INK4a and p14^ARF protein expression between GCA and GDAObjective: To explore the possible difference of p16^INK4a and p14^ARF expression between GCA and GDA.Methods: Forty-four samples from cardia and 40 tumors from distal schomach were chosen by the procedure above mentioned. Meanwhile, twenty-five samples with normal gastric mucosa were used as control group, obtained from gastric stump resected at the corresponding time period. Expressions of p16^INK4a and p14^ARF were detected in two subtypic tumors by immunohistochemistry.Results:2.1 Expression of p14^ARF in gastric tumors2.1.1 The expression of p14^ARF in GCA and GDAP14^ARF immunoreaction was located in cell nucleus and/or cytoplasm. The values of p14^ARF expression were 84.0% and 45.2% in normal gastric mucosa and tumors, respectivly, showing significantly statistics （P<0.05）. Moreover, expression of p14^ARF was lower in GCA than that in GDA （34.1% vs 57.5%, P<0.05）.2.1.2 Relationship between p14^ARF and clinical pathological characters in GCA and GDAThere was no relationship between p14^ARF protein expression and the sex, age, histological differentiation and lymph node metastasis of the patients with GDA （P>0.05）. Wherease, it appeared that p14^ARF protein is related with the differentiation and lymph node metastasis in GCA. Only 18.5% of GCA with poor differetiation showed positve reaction, which was significantly lower than that in well differented tumors （58.8%, P<0.05）. And also the p14^ARF expression was lower in tumors with lymph node metastasis than that without metastasis （17.4% vs 52.4%, P<0.05）. No relationship between p14^ARF and sex, age in cardiac tumors was found （P>0.05）.Moreover, p14^ARF expression displayed markedly difference in the GCA and GDA subgroup with poor differentiation. GDA with poor differentiation showed higher reaction than cardiac ones （55.2% vs 18.5%, P<0.05）. And the expression of p14^ARF protein was statisticly downregulated in GCA with lymph node metastasis when compared with the GDA with lymph node metastasis （17.4% vs 57.1%, P<0.05）.2.2 Expression of p16^INK4a in gastric tumors2.1.1 The expression of p16^INK4a in GCA and GDAImmunohistochemisty results showed that only 58.3% gastric carcinomas displayed positive reaction by p16^INK4a antigen, which was markedly lower than normal gastric mucosa （96.0%, P<0.05）. Furthermore, expression of p16^INK4a was significantly lower in GCA than that in GDA （43.2% vs 75.0%, P<0.05）.2.2.2 Relationship between p16^INK4a and clinical pathological characters in GCA and GDAThere were no relationships between p16^INK4a expression and the age, sex, hitological differentiation and lymph node metastasis of the patients with GCA and GDA （P>0.05）. However, the different expression of p16^INK4a in two subtypic tumors with poor differentiation was detected, and the values were 37.0% and 72.4% in GCA and GDA with poor differentiation, respectively. Meanwhile, p16^INK4a expression was lower in GCA with lymph node metastasis than that in GDA with lymph node metastasis （39.1% vs 75.0%, P<0.05）.PartⅢThe difference of p16^INK4a and p14^ARF gene deletion between GCA and GDAObjective: To explore the possible differences of p16^INK4a and p14^ARF gene deletions between GCA and GDA.Methods: Forty-four samples from cardia and 40 tumors from distal schomach were chosen by the procedure above mentioned. Meanwhile, fifteen samples with normal gastric mucosa were used as control group, obtained from gastric stump resected at the corresponding time period. Deletions of p16^INK4a （E1αand E2） and p14^ARF （E1βand E2） were detected in two subtypic tumors by PCR assay.Results:3.1 Comparative study of p16^INK4a gene deletion in E1αand E2 exon between GCA and GDA.PCR results showed that there was no deletion of E1αand E2 exon in normal gastric mucosa. While in 84 cases of gastric carcinomas, E1α, E2 exon deletion was seen in 9.5% （8/84） and 8.3% （7/84） cases, respectively, and the total deletion proportion of p16^INK4a gene in E1αand E2 exon was 17.9% （15/84） in gastric carcinomas.In 44 cases of GCA, deletion of E1α, E2 and the E1αcombined with E2 was 4 cases （9.1%）, 5 cases （11.4%） and 9 cases （20.5%）, respectively. In 40 cases of GDA, deletion of E1α, E2 was 10.0% （4/40） and 5.0% （2/40）, respectively, and p16^INK4a （E1αand E2） deletion was 15.0% （6/40）. There was no significant differences on E1α, E2 and p16^INK4a （E1αand E2） between GCA and GDA （P>0.05）.3.2 Comparative study of p14^ARF gene deletion in E1βand E2 exon between GCA and GDA.PCR results showed that there is no deletion of E1βand E2 exon in normal gastric mucosa. In 84 cases of gastric carcinomas, deletion of E1β, E2 could be seen in 6.0% （5/84） and 8.3% （7/84） cases, respectively, and otal deletion proportion of p14^ARF （E1βand E2） was 14.3% （12/84）.In 44 cases of GCA, deletion of E1β, E2 and E1βcombined with E2 was 3 cases （6.8%）, 5 cases （11.4%） and 18.2% cases （8/44）, respectively. Besides, in 40 cases of GDA, deletion of E1β, E2 was 5.0% （2/40） and 5.0% （2/40）, respectively, and p16^INK4a （E1αand E2） deletion was 10.0% （4/40）. There was no significant differences on E1α, E2 and p14^ARF （E1βand E2） between GCA and GDA （P>0.05）.3.3 Relationship of p14^ARF and p16^INK4a expressions and gene homozygous deletionIn 84 cases of gastric carcinomas, 35 out of 84 cases displayed negative expression of p16^INK4a, and 15 out of 84 cases showed p16^INK4a gene deletion in E1αand E2 exon. Simultaneously, the p16^INK4a protein expression was negative among all the 15 cases with p16^INK4a gene deletion. And in all cases, negative expression of p14^ARF was shown in 46 cases. The p14^ARF gene deletion （E1βand E2） was only seen in 12 cases, accompanying by negative immunoreaction of p14^ARF antigen. The results may suggest that deletion of p14^ARF and p16^INK4a was one of the mechanisms leading to protein inactivation.PartⅣCompasion of p16^INK4a and p14^ARF methylation between GCA and GDAObjective: To explore the possible difference of p16^INK4a and p14^ARF methylation between gastric cardiac and distal adenocarcinomas, and study the mechanism of p16^INK4a and p14^ARF inactivation in gastric tumors.Methods: Fifty-two cardiac adenocarcinomas, and 39 distal ones and 15 cases of normal gastric mucosa were detected by Methylmion Specific PCR （MSP） for p16^INK4a and p14^ARF methylation. Meanwhile, 10 cases of tumors from cardia and 8 cases from distal stomach were used to total RNA obtaining to detect expressions of p16^INK4a and p14^ARF at mRNA level by Real-time PCR. Results:4.1 p14^ARF methylation by MSP4.1.1 Comparative study of p14^ARF gene methylation between GCA and GDAMSP result showed that the frequency of p14^ARF methylation in gastric tumors was markedly higher than that in normal gastric mucosa （56.0% vs 13.3%, P<0.05）. Hypemethylation of p14^ARF was 61.5% and 43.6% in GCA and GDA, respectively, which was higher than that in normal gastric mucosa （P<0.05）. Moreover, p14^ARF hypemethylation in cardiac carcinomas was higher than in distal ones （P<0.05）.4.1.2 Relationship of p14^ARF hypermethylation with clinical pathological characters between GCA and GDAP14^ARF methylation was not associated with the age, sex, histological differentiation and lymph node metastasis of the patients with GDA （P>0.05）. P14^ARF methylation in GCA with poor differentiation was higher than that with well differentiation （78.1% vs 45.5%, P<0.05）, but there was no relationship between p14^ARF methylation and sex, age and lymph node metastasis in GCA （P>0.05）. Furthermore, different frequency of p14^ARF methylation between GCA and GDA with poor differentiation was detected （78.1% vs 48.0%, P<0.05）.4.2 Methylation of p16^INK4a by MSP4.2.1 Comparative study of p16^INK4a methylation between GCA and GDAPromoter methylation was detected in 63.7% of gastric tumors, higher than that of normal mucosa （20.0%, P<0.05）. Methylation of p16^INK4a was 73.1% and 51.3% in gastric cardiac and distal adenocarcinomas, respectively, and promoter methylation was more frenquency in GCA than distal ones （P<0.05）.4.2.2 Relationship of p14^ARF hyermethylation with clinical pathological characters between GCA and GDANo correlation was found between promoter hyermethylation and clinical pathological parameters in both tumors from two sites （P>0.05）. However, promoter hypermethylation of p14^ARF was detected in 81.3% of cardiac tumors and in 56.0% of distal ones with poor differentiation, and the former was higher than the latter （P<0.05）.4.3 Effection of promoter hypermethylation on mRNA expression of p14^ARF and p16^INK4a in gastric tumorsEighteen fresh samples of gastric carcinomas were selected, including 10 of cardiac carcinomas and 8 of distal ones. And they were classified two groups according to methylation status. Among 18 cases, 10 cases were positive regarding of p14^ARF methylation, 8 cases were negative. According to p16^INK4a methylation, half of cases were positive. Expression of p14^ARF and p16^INK4a at mRNA level were detected by Real-time PCR further, and the result showed that expression of p14^ARF and p16^INK4a in hypermethylated group was significantly lower when compared with that in unmethylated group （P<0.05）. It may indicate that methylation of p14^ARF and p16^INK4a genes could inhibit the expression of p14^ARF and p16^INK4a at mRNA level.Conclusion:1 Higher expression of CK14 and lower expression of MUC2 in gastric cardiac carcinomas than in distal ones may indicate that GCA is different from GDA in histotype and histological origin.2 The expression of p14^ARF and p16^INK4a protein was significant lower in GCA that in GDA, and p14^ARF expression was related with differentiation and lymph node metastasis in GCA. However, the expression of both p14^ARF and p16^INK4a protein was not associated with differentiation and lymph node metastasis in GDA. Therefore, there were differences in expression of p14^ARF and p16^INK4a and its clinical pathlogical significance between GCA and GDA.3 Expressions of p16^INK4a and p14^ARF protein were negative in the cases with p16^INK4a and p14^ARF gene deletion. However, there was no significant difference in the occurance of deletions of p16^INK4a and p14^ARF gene between GCA and GDA, suggesting that there might be no correlation between deletion of p16^INK4a and p14^ARF gene and subsites of gastric carcinomas.4 Methylation levels of both p14^ARF and p16^INK4a were higher in GCA than that in GDA. The differences in the methylation level may be the important contributing factor to the differences in the mRNA and protein between cardiac adenocarcinoma and distal gastric adenocarcinoma.更多还原