【作者】 张修礼；

【导师】 李兆申； 徐东平； 孙自勤；

【作者基本信息】 第二军医大学 ， 消化内科学， 2007， 博士

【摘要】 背景过去的20年,胃癌在病因学、发病机制及治疗学上均取得了巨大进步。特别是随着更多癌基因和抑癌基因的不断发现,人们对胃癌病理机制的认识也不断深化和逐步清晰;如CDH1基因和表皮生长因子受体(EGFR)家族在胃癌发病机制中的作用被人们发现和认识。随之而来的是治疗学上的不断创新和突破(如针对ERBB2的人源性单抗Herceptin已用于伴有ERBB2过表达的肿瘤病人的治疗)。此外,不断出现的新的研究方法也为我们进一步揭示胃癌的病理机制提供了有力支持,如基因芯片技术的“高通量”特性为我们深入探讨肿瘤发病过程中的“多分子变化”提供了重要手段。尽管ERBB2在肿瘤病理机制中的研究比较深入,但在胃癌的研究方面国内还缺乏对它的系统研究;加之近年来“ERBB2、ERBB1酪氨酸激酶编码区基因突变”的研究成为该领域研究的热点,国内却未见相关的研究报道。基于此,我们利用基因芯片技术,对2例胃癌(lauren分类:肠型胃癌)的癌组织及其癌旁组织表达谱的基因学变化进行了观察;在此基础上,我们选取基因芯片被证实上调表达的重要的癌基因-EGFR受体家族中的ERBB2和ERBB3(ratio分别为:7.8,2.6)进行了免疫组织化学分析(IHC);并检测了ERBB2 mRNA水平的表达;同时,在基因组水平对ERBB2酪氨酸激酶编码区基因变异进行了观察。本文旨在进一步阐述胃癌病理过程中基因表达方面的差异、深入探讨癌基因-ERBB2、ERBB3在胃癌发病过程中的作用,并试图揭示我国胃癌病人是否也存在基因组水平ERBB2酪氨酸激酶编码区基因突变。方法①选取2例新鲜胃癌组织(lauren分类:肠型胃癌)及其对应的癌旁组织进行表达谱的基因芯片分析。②利用免疫组织化学方法对65例胃癌及25例癌旁正常组织的ERBB2和ERBB3在蛋白水平的表达进行了观察,并与临床资料进行了比较分析。③利用real-time RT-PCR对12例新鲜配对胃癌和癌旁组织中ERBB2 mRNA表达情况进行了相对定量,对比分析其mRNA表达。④对55例胃癌及25例癌旁组织提取基因组,利用套式PCR方法扩增ERBB2酪氨酸激酶编码区(18-23外显子),对PCR产物进行了测序、序列比对,观察其是否存在酪氨酸激酶编码区基因变异。结果1. 14,000点基因芯片实验结果:2例肠型胃癌上调表达的基因分别有1101条、986条;下调表达的基因分别为762条、553条。2例胃癌共同上调表达的基因有154条,共同下调表达的基因有79条。上调表达基因中包括了癌基因(如ERBB2、ERBB3等)、细胞周期调节蛋白(如cyclinD1、cyclinB1)、细胞代谢类分子(如CYP3A5、CYP2C8等)、上皮细胞分类化分子(如PGC、MUC1、MUC6等)及其他类分子。2. 65例胃癌及25例癌旁组织的ERBB2和ERBB3的免疫组织化学观察:胃癌组织中ERBB2和ERBB3表达阳性率分别为12.3%和10.8%,两者阳性分布区域相同,均定位于胞膜,癌旁组织均为阴性表达。其中ERBB2和ERBB3共表达2例,占3.1%。且ERBB2在肠型胃癌的阳性率高于弥散型(16.3% vs 4.5%,P<0.05),而ERBB3在弥散型胃癌的表达阳性率高于肠型胃癌(27.6% vs 2.3%,P<0.05)。3.实时定量RT-PCR结果:胃癌组织与癌旁组织中ERBB2 mRNA的表达存在差异,12例胃癌中8例ERBB2 mRNA的表达阳性(8/12),而12例癌旁组织中ERBB2 mRNA的表达阳性(3/12),两者相差显著(66.7% vs 25.0%,P<0.01)。进一步的Ct值分析表明,与癌旁组织相比,胃癌组织ERBB2表达平均增高5.76倍(P<0.01)。4.套式PCR及DNA测序:55例胃癌及25例正常胃组织均获得了ERBB2酪氨酸激酶编码区18-23外显子的扩增,并进行了测序。在胃癌组织中仅发现了1例TGC-TCC错义突变,且存在于其内含子内,ERBB2胞内段的酪氨酸激酶编码区(18-23外显子)未发现基因变异;所有癌旁组织均无ERBB2基因突变。结论1.对2例肠型胃癌进行的基因芯片的表达谱分析结果表明,与癌旁组织相比,胃癌组织内基因表达产生了严重紊乱,多种基因表达得以上调和下调,部分癌基因被活化。这些基因影响到细胞的生长、代谢、增生、细胞周期及细胞间信号传导等多个方面。2. ERBB2蛋白和ERBB3蛋白在胃癌组织中均存在过表达,且ERBB2更多见于肠型胃癌,而ERBB3则更多见于弥散型胃癌。提示作为受体酪氨酸激酶重要成员的ERBB2和ERBB3可能在不同病理类型的胃癌中发挥不同的作用。3.癌基因ERBB2在胃癌组织中存在明显的转录水平(mRNA)的活化。4.55例胃癌基因组水平未发现ERBB2酪氨酸激酶区的基因突变,提示我国胃癌患者ERBB2酪氨酸激酶编码区的基因突变率低,最少不是胃癌发生过程中的“频发事件”。更多还原

【Abstract】 Background In the past two decades many important dicoveries and improvements have been made in the gastric cancer research regarding the etiology, pathogenesis and therapeutic methods. Especially with the dicovery of more and more oncogenes and tumor reppressor genes, for example, the identification of CDH1 and human epithelial growth factor receptors family, the carcinogenesis of gastric cancer is becoming more and more clear, and our insight into gastric cancer is deepening step by step. Subsequently, more new innovations and even some breakthrough were made therapeutically,and herceptin- a humanized monoclonal antibody used in the gastric cancer patient with ERBB2 over expression, is the case. Additionally, the development of new approaches to functional genomics, for example the characteristic of“high output”of microarray, has markedly improved our ability to explore molecular alterations underlying gastric carcinogenesis and progression.In the first part of our article, we screened the differentially expressed genes by studing two cases of intestinal type of gastic cancer tissue using cDNA microarray compared with the adjacent normal part. Subsequently immunohistochemistry (IHC) was appllied to investigate the expression of ERBB2 and ERBB3- the up regulated oncogenes which have been identified in the first part. At the same time, real-time quantitative reverse transcriptional ploymerase chain reaction was performed to quantify the relative expression of mRNA of ERBB2 in the gastric cancer tissue. And the genomic mutations of ERBB2 coding sequence of tyrosine kinase were detected by nested PCR and then sequenced. The aim of the study is to elicit the carcinogenesis of the receptor tyrosine kinases (especially ERBB2 and ERBB3) in the gastric cancer, and ascertain whether there exist genomic mutations in the coding sequence of ERBB2 tyrosine kinases. Methods Two pairs of fresh resected samples (intestinal type of gastric caner tissue and the matched adjacent normal tissue according to the Lauren classfication) were appllied to cDNA microarray to screen the differentially expressed genes. The expression of ERBB2 and ERBB3 which were confirmed to be over-expressed in the microarray test, also known as the important members of epithelial growth factor receptor(EGFR) family, were detected in 65 cases of gastric cancer and 25 cases of adjacent normal tissue using immunohistochemistry (IHC). The results of expression of ERBB2 and ERBB3 in gastric cancer tissue were compared with the clinical parameters. Relative quantification of mRNA of ERBB2 in 12 pairs of fresh specimens (the gastric cancer tissue and the matched adjacent noraml tissue) was quantified by real-time reverse transcriptional polymerase chain reaction. The genome was extracted from 55 gastric caner tissues and 25 adjacent normal tissues, and the coding sequence of tyrosine kinase of ERBB2 was amplified by nested-PCR. The PCR products were sent to be sequenced.Results1. The results of gene expression profile by cDNA microarray analysis (1,4000 unique human cDNA sequences): Over 1000 genes were over-expressed in the 2 cases, and 762 genes and 553 genes were under-expressed respectively. Among the two cases, 154 genes were identified as the co-over-expressed genes and 79 genes as the co-under-expressed genes. Oncogenes such as ERBB2 and ERBB3,cell cycle regulating proteins such as cyclinD1 and cyclinB1, molecules of cell metabolism such as CYP3A5、CYP2C8, and cell epithelial cluster antigens such as mucin 1 and mucin 6,were identified.2. IHC demonstrated that ERBB2 and ERBB3 were over-expressed, accounting for 12.3% and 10.8% respectively in the gastric cancer tissues, while there was no positive stain in the adjacent normal tissues. The localization of ERBB2/ERBB3 was mainly the cytomembrane and/or cytoplasma. There were two cases of co-over-expressed ERBB2 and ERBB3, accounting for 3.3%. Furthermore, the rate of positive stain of ERBB2 in the intestinal type of gastric cancer is higher than that in the diffuse type; while the expression of ERBB3 demonstrated the opposite results.3. The results of real-time RT-PCR: The significant difference of the expression of mRNA between cancer tissue and the matched adjacent normal tissue was observed. Of the 12 gastric cancer cases, eight were postive for mRNA of ERBB2;while only three were positive in the 12 adjacent normal tissues. There was 5.76 folds elevation of ERBB2 mRNA in gastric cancer tissue than that in the adjacent tissue by evaluation of Ct. 4. Results of amplification of coding sequence of tyrosine kinase of ERBB2 by nested PCR: Fifty-five cases of cancer tissue got the complete amplification of all the exons of ERBB2 tyrosine kinase for sequencing, and twenty five cases of normal tissue got the complete amplification. No mutations were found in the coding sequence of tyrosine kinase of ERBB2 both in cancer and normal tissues except one missense mutation of TGC-TCC in the intron.Conclusions1. Severe disturbance of genes expression profile in gastric cancer is a common phenomenon. The over-expressed and under-expressed genes extensively and intensively affect cell biocycle, especially the cell growth, proliferation, metabolism, signal communication and so on.2. The different expresson of ERBB2 and ERBB3 detected by IHC in our study suggested that ERBB2 and ERBB3 may play different role in the gastric carcinogenesis of the two gastric cancer types(intestinal and diffuse type according to Lauren’s classfication).3. There exists marked activation of ERBB2 in the transcriptive process in gastric cancer tissue than that of matched adjacent normal tissue.4. The analysis of genomic mutation of coding sequence of tyrosine kinase of ERBB2 implied that the genomic mutation of ERBB2 is not common in Chinese gastric cancer patients, or that is at least not a“high molecular incidence”in Chinese gastric cancer patients. Our results seemed to differ from the reports of abroad published papers.更多还原