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妾崎岑
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1 学校编码 :10384 密级学号 : 硕士学位论文诱骗受体 3 与细胞外信号调节激酶 1/2 在胃癌发生发展中的相关性研究 A Study on mechanism of Decoy Receptor 3 and Extracellular-signal-regulated-kinase 1/2 in Gastric Cancer 指导教师姓名 : 杨东海刘忠臣庄国洪副教授专业名称 : 外科学论文提交日期 : 2010 年 4 月论文答辩日期 : 2010 年 6 月 2010 年 4 月

3 厦门大学学位论文原创性声明本人呈交的学位论文是本人在导师指导下, 独立完成的研究成果本人在论文写作中参考其他个人或集体已经发表的研究成果, 均在文中以适当方式明确标明, 并符合法律规范和厦门大学研究生学术活动规范 ( 试行 ) 另外, 该学位论文为 ( ) 课题 ( 组 ) 的研究成果, 获得 ( 资助, 在 ( ) 课题 ( 组 ) 经费或实验室的 ) 实验室完成 ( 请在以上括号内填写课题或课题组负责人或实验室名称, 未有此项声明内容的, 可以不作特别声明 ) 声明人 ( 签名 ): 年月日

4 厦门大学学位论文著作权使用声明本人同意厦门大学根据中华人民共和国学位条例暂行实施办法等规定保留和使用此学位论文, 并向主管部门或其指定机构送交学位论文 ( 包括纸质版和电子版 ), 允许学位论文进入厦门大学图书馆及其数据库被查阅借阅本人同意厦门大学将学位论文加入全国博士硕士学位论文共建单位数据库进行检索, 将学位论文的标题和摘要汇编出版, 采用影印缩印或者其它方式合理复制学位论文本学位论文属于 : ( )1. 经厦门大学保密委员会审查核定的保密学位论文, 于年月日解密, 解密后适用上述授权 ( )2. 不保密, 适用上述授权 ( 请在以上相应括号内打或填上相应内容保密学位论文应是已经厦门大学保密委员会审定过的学位论文, 未经厦门大学保密委员会审定的学位论文均为公开学位论文此声明栏不填写的, 默认为公开学位论文, 均适用上述授权 ) 声明人 ( 签名 ): 年月日

5 摘要胃癌是我国常见的恶性肿瘤之一, 是最常见的消化道恶性肿瘤早期胃癌多无症状或仅有轻微症状当有明显不适就医时, 往往病变已属晚期虽然胃癌发病的确切机制尚不完全清楚, 但是环境因素遗传因素等调节机制在胃癌的癌前病变及肿瘤发生发展过程中均处于重要的地位, 免疫调节在胃癌的发病过程中也起着重要的作用肿瘤坏死因子 (tumor necrosis factor,tnf) 家族能够调节多种生理活动, 例如 : 细胞生长分化, 细胞存活凋亡, 产生细胞因子以及淋巴细胞的共刺激作用等等 TNFR 家族有四种诱骗受体 :DcR1 DcR2 DcR3 和 OPG DcR3 又称 TR6, 是新近发现的一个新的 TNFR 超家族成员它能竞争性地与 LIGHT 及 FasL 结合并抑制其介导的凋亡研究发现 DcR3 在多种肿瘤细胞和某些自身免疫病中高度表达, 可以显著上调肿瘤发生的几率, 可以促进血管生成, 诱导树突状细胞凋亡, 抑制巨噬细胞分化等细胞外信号调节激酶 (Extracellular-signal-regulated kinase 1/2,ERK1/2, p44/p42) 是有丝分裂原活化蛋白激酶 (mitogen-activated protein kinase,mapk) 家族的重要成员两者广泛地存在于人体各类细胞中通过 Ras/Raf/MAPKKK/MAPKK/MAPK/ERK 通路,Ras 等基因受刺激后, 活化 MAPK ERK1/2 等通路, 促进细胞的增殖分化近期的研究表明,ERK1/2 的高表达, 对乳腺癌大肠癌胰腺癌胆管癌恶性黑色素瘤白血病及粘液瘤等肿瘤, 皆可作为独立的预测患者预后情况的参考指标为了研究 DcR3 及 ERK1/2 在胃癌发生发展中的作用及其免疫机制, 本实验首先对临床获取的胃癌肿瘤病人手术切除的组织进行了检测, 利用 RT-PCR 检测肿瘤组织中 DcR3 及 ERK1/2 mrna 水平的表达, 以 Western blot 技术分析肿瘤组织中 DcR3 及 ERK1/2 蛋白水平的表达情况, 并进一步利用免疫组织化学方法了解 DcR3 及 ERK1/2 在肿瘤组织中的定位情况获得了临床胃癌病人肿瘤组织中 DcR3 及 ERK1/2 表达的基本情况后, 采用人胃癌细胞系 BGC823 肌肉注射的方法建立了胃癌裸鼠肿瘤模型, 观察裸鼠肿瘤各组织器官 I

6 的影响变化, 并利用 RT-PCR Western blot 免疫组化等技术检测裸鼠肿瘤组织和心脏肝脏等各器官中 DcR3 及 ERK1/2 mrna 水平蛋白水平的表达情况及其在组织中的定位情况实验发现胃癌患者肿瘤组织中 DcR3 和 ERK1/2 mrna 水平和蛋白水平的表达量都较癌旁组织有明显上升 ; 利用人胃癌细胞系 BGC823 建立的胃癌裸鼠模型的肿瘤组织中 DcR3 和 ERK1/2 也出现高表达比较后可发现在对胃癌动物模型和临床胃癌患者肿瘤组织的检测中,DcR3 和 ERK1/2 在肿瘤的发生发展过程中均呈过表达趋势, 具有协同表达的特点因此, 在胃癌的发生发展过程中,DcR3 和 ERK1/2 的表达存在密切联系, 进一步研究可对于临床胃癌的早期诊断和免疫治疗提出一种新的思路关键词 : 胃癌诱骗受体 3 细胞外信号调节激酶免疫治疗 II

7 Abstract Gastric cancer is one of common malignant tumors in China, is the most common gastrointestinal malignancy. Early gastric cancer always with only mild even no symptoms, when there is significant symptoms, disease is already advanced. Although the precise pathogenesis of gastric cancer is remains unclear, environmental factors, genetic factors were central to the mechanism of gastric precancerous lesions and tumor development, the immune regulation also plays an important role in the disease progression. Tumor necrosis factor (TNF) family can regulate multiple physiological activities, such as: cell growth, differentiation, cell survival, apoptosis, cytokine production and lymphocyte co-stimulation, etc. TNFR family has four decoy receptors: DcR1, DcR2, DcR3 and OPG. DcR3, also known as TR6, is a member of TNFR superfamily which is newly identified. It could binding with LIGHT or FasL and inhibit LIGHT or FasL-mediated apoptosis. Recent studies found that DcR3 could be found highly expressed in a variety of tumors and certain autoimmune diseases which would promote angiogenesis, induce apoptosis of dendritic cells, inhibit macrophage differentiation. Extracellular signal-regulated kinase (ERK1/2, p44/p42) is members of a mitogen-activated protein kinase ( MAPK) family, widely exist in the human body. Through the Ras / Raf / MAPKKK / MAPKK / MAPK / ERK pathway, Ras and other genes affected by stimulation, activation of MAPK, ERK1/2 and other pathways to promote cell proliferation and differentiation. Recent studies show that, ERK1/2 were highly expression in breast cancer, colorectal cancer, pancreatic cancer, bile duct cancer, malignant melanoma, leukemia and tumors such as myxoma, which may apply as a separate forecast of prognosis in patients with a reference index. In order to investigate the effect and immune mechanism that DcR3 and ERK1/2 in gastric cancer occur and development, I adopted measures as follow: first, I collected 50 clinical gastric cancer patients tumor tissue, detected expression of DcR3 and III

8 ERK1/2 in mrna level with RT-PCR and Western blot analysis of which expressed in protein level, using immunohistochemistry to understand localization of DcR3 and ERK1/2 in the tumor tissue. After that, we established nude rats gastric cancer animal model by injecting human gastric cancer cell lines BGC823, using RT-PCR, Western blot, immunohistochemistry to detect expression of DcR3 and ERK1/2 in tumor tissue of nude mice and rats heart, liver, spleen, lung, kidney and its position in the organization situation. During the research, I found that expression of DcR3 and ERK1/2 were significantly higher than the adjacent tissues in patients with gastric cancer. The tumor of animal models which were established with human gastric cancer cell line BGC823 also appears highly expression of DcR3 and ERK1/2. DcR3 and ERK1/2 in the process of tumor development showed overexpression tendency, has coordinated characteristics. Therefore, in the process of development of gastric cancer, DcR3 and ERK1/2 expressed closely. Further studies may provide a new diagnosis method or immune therapy idea to cure gastric cancer. Keywords:Gastric cancer; DcR3; ERK1/2; Immune therapy IV

9 目录摘要...I ABSTRACT...III 第一章前言...1 一胃癌的发病因素环境因素遗传因素免疫因素癌前期变化胃的癌前期状态胃的癌前期病变...2 二胃癌的相关临床表现胃癌各期症状胃癌病人体征胃癌常见并发症...4 三临床上常用的胃癌分型方法根据胃癌的发生部位分型根据具体形态分型早期胃癌中晚期胃癌根据胃癌组织分型根据组织结构分型 : 根据组织发生方面分型 :...5 四临床常用的胃癌分期方法 UICC/TNM 分期法 JGCA 分期法...6 五胃癌的诊断 :...7 V

10 1 实验室检查 X 线表现纤维内窥镜检查脱落细胞学检查 B 超 CT 检查胃癌的 X 线表现早期胃癌的 X 线表现中晚期胃癌的 X 线表现内镜检查早期胃癌中晚期胃癌胃液检查生物学与生物化学检查...9 六胃癌的治疗手术治疗根治性切除术姑息性切除术短路手术放射治疗化学治疗中医治疗免疫治疗中药治疗具有较强的整体观念可以弥补手术治疗放射治疗化学治疗的不足不影响劳动能力副作用小...12 七肿瘤发生发展的信号调控 DcR3 与肿瘤 DcR3 与 TNF DcR3 与 Fas/FasL...18 VI

11 7.1.4 DcR3 与 T 细胞 DcR3 与免疫调节 ERK/MAPK 信号通路 ERK 与肿瘤 ERK 在其他系统中的作用 DcR3 与 ERK...26 八实验方案设计研究目标研究内容 DcR3 在肿瘤发生发展中的表达特点 ERK1/2 调控 DcR3 的分子机制探讨...28 第二章胃癌患者肿瘤组织中 DCR3 及 ERK1/2 的表达情况研究及临床分析...30 一材料和方法材料实验标本收集主要试剂及耗材主要仪器及设备主要溶液的配制方法 RT-PCR 法检测胃癌患者肿瘤组织中 DcR3 及 ERK1/2 mrna 表达情况胃癌肿瘤组织中总蛋白的提取 Western blot 方法测定胃癌患者肿瘤组织中 DcR3 及 ERK1/2 蛋白表达情况免疫组织化学方法测定胃癌患者肿瘤组织中 DcR3 及 ERK1/2 定位情况统计学方法处理实验数据...37 二结果与分析 RT-PCR 方法检测胃癌患者肿瘤组织中 DcR3 及 ERK1/2 mrna 表达情况 Western-blot 方法检测胃癌患者肿瘤组织中 DcR3 及 ERK1/2 蛋白表达 VII

12 免疫组织化学法检测胃癌患者肿瘤组织中 DcR3 ERK1/2 定位情况综合检测结果...41 三讨论...45 第三章胃癌裸鼠模型的建立和 DCR3 ERK1/2 在胃癌细胞株及裸鼠模型肿瘤组织各器官中的表达情况研究...47 一材料和方法材料实验裸鼠及细胞株主要试剂及耗材主要仪器及设备主要溶液的配制方法人胃癌细胞株 BGC823 培养胃癌裸鼠模型的建立及分组 RT-PCR 检测 BGC823 细胞胃癌裸鼠肿瘤细胞及各组织器官中 DcR3 ERK1/2 mrna 的表达胃癌肿瘤细胞 BGC823 中总蛋白的提取胃癌裸鼠肿瘤及各组织器官总蛋白的提取 Western blot 检测胃癌裸鼠肿瘤组织及各组织器官中 DcR3 ERK1/2 蛋白的表达免疫组织化学方法测定胃癌裸鼠肿瘤组织及各组织器官中 DcR3 ERK1/2 定位情况统计学数据处理...56 二结果与分析人胃癌细胞株 BGC823 对裸鼠的影响 RT-PCR 方法检测胃癌细胞株 BGC823 裸鼠肿瘤组织及各组织器官中 DcR3 ERK1/2 mrna 的表达 Western-blot 检测人胃癌肿瘤细胞及胃癌裸鼠肿瘤组织及各组织器官中 DcR3 及 ERK1/2 蛋白的表达免疫组织化学法检测胃癌裸鼠肿瘤组织及各组织器官中 DcR3 ERK1/2 的定位情况...60 三讨论...62 VIII

13 总结...65 参考文献...66 致谢...74 IX

14 CONTENTS ABSTRACT...III CHAPTER I INTRODUCTION...1 I ETIOLOGY OF GASTRIC CANCER Environmental factors Hereditary factors Immunity factors Precancerous lesion Precancerous conditions Precancerous lesions...2 II CLINICAL MANIFESTATIONS OF GASTRIC CANCER Symptoms Physical examinations Common complications...4 III CLINICAL TYPING OF GASTRIC CANCER Locations Figures Early stage Advanced stage Tissue typing Tissue structure Tissue originate...5 Ⅳ CLINICAL STAGING OF GASTRIC CANCER UICC/TNM JGCA...6 Ⅴ DIAGNOSIS OF GASTRIC CANCER Laboratory examination X-ray...7 X

15 3 Fiber endoscopy Cytology examination Type-B ultrasonic CT Results of X-ray Early stage Advanced stage Fiber endoscopy Early stage Advenced stage Gastric juice examination Biologic and biochemical examination...9 Ⅵ TREATMENT OF GASTRIC CANCER Operation Radical operation Palliative operation By-pass operation Radiation therapy Chemiotherapy Chinese traditional therapy Immunotherapy Traditional Chinese medicine therapy More concept of wholism Cover the shortage of other treatments No influence of labor capacity Less side effect...12 Ⅶ SIGNAL CONTROL OF TUMORIGENESIS DcR3 and tumor DcR3 and TNF DcR3 and Fas/FasL DcR3 and T cells DcR3 and immunoregulation...20 XI

16 7.2.1 ERK/MAPK signal passway ERK and tumor ERK in other system DcR3 与 ERK...26 Ⅷ EXPERIMENT DESIGN Objective and significance Contents Exprssion character of DcR Molecular mechanism of ERK1/ CHAPTER II ANALYSIS OF EXPRESSION OF DCR3 AND ERK1/2 IN PATIENTS` TUMOR TISSUES...30 I MATERIALS AND METHODS Materials Samples of human tumor and normal tissues and cell lines Main reagents and consumptive material Primary instruments and equipments Primary solution making Methods Exprssion of DcR3 and ERK1/2 mrna in human tumor and normal tissues detect by RT-PCR Extraction of total protein of human tumor tissues Expression of DcR3 and ERK1/2 protein in human tumor and normal tissues detect by Western blot Location of DcR3 and ERK1/2 in human tumor tissues detect by immunohisochemistry Statistical method to deal with experimental datas...37 II RESULTS AND ANALYSIS Expression of DcR3 and ERK1/2 mrna in human tumor and normal tissues detect by RT-PCR Expression of DcR3 and ERK1/2 protein in human tumor and normal tissues detect by Western blot Location of DcR3 and ERK1/2 in human tumor tissues detect by immunohisochemistry...40 XII

17 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

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