【作者】 龚道军；

【导师】 陈力；

【作者基本信息】 浙江大学 ， 普通外科疾病的基础与临床， 2009， 博士

【摘要】 前言2007年,在世界范围内,胃癌仍然是排在第四位的常见恶性肿瘤,估计有100万的新发病例,其中将近70%发生在发展中国家。通常,在亚洲、部分南美国家和一些非洲国家,胃癌的发病率是最高的。在男人和女人的疾病死亡原因中,胃癌分别排在第二位和第四位。2007年,全球范围内大约有80万人死于胃癌。我国胃癌平均年死亡率约为25.2/10万人口。目前,胃癌的主要治疗措施是手术、化疗和放疗,通常是两种或多种方法相结合。但是,除非早期发现,胃癌难以治愈。在美国,胃癌的5年生存率为24%,如果早期发现,将会提高到61%。采用两种或几种标志物联合检测,有利于提高胃癌的早期诊断率,增进疗效、判断预后和检出复发。目前,CEA和CAl9-9是胃癌的两个主要的肿瘤标志物。发现新的肿瘤标记物是肿瘤的研究方向之一。CD97分子是表皮生长因子二类七次跨膜受体(EGF-TM7)家族成员之一,拥有独特的杂交结构,N-末端含有3个表皮生长因子(EGF)样功能区,通过粘蛋白样茎区与七次跨膜受体(TM7)偶联。在正常组织中,除小神经胶质细胞以外,CD97只能在巨噬细胞和树突状细胞,以及一些T、B淋巴细胞中丰富表达。如今,CD97被发现在一些上皮性癌组织中丰富表达,如甲状腺、胃肠、胰腺导管和口腔鳞状细胞癌。不同的证据表明,CD97在肿瘤的去分化、迁移、浸润和转移中发挥重要作用。然而,CD97对胃癌细胞生物学特性的影响及其内在机制仍然有待于阐明。第一章CD97-siRNA转染对人胃癌细胞株MKN45增殖、凋亡、迁移及侵袭能力的影响目的:设计合成有效的CD97-siRNA,体外转染胃癌细胞株,观察CD97表达变化及其对体内、外胃癌细胞增殖、凋亡、迁移及侵袭能力的影响。方法:1.采用人胃癌细胞株MKN45,针对CD97基因设计siRNA,采用化学法合成,筛选出最有效的CD97-siRNA。2.CD97-siRNA转染胃癌细胞48小时后,用RT-PCR检测CD97基因在mRNA水平表达的变化,72小时后用Western blot和免疫细胞化学方法检测CD97基因在蛋白水平表达的变化,并用MTT法和流式细胞术检测胃癌细胞增殖、凋亡的变化,用细胞迁移和侵袭试验检测胃癌细胞迁移和侵袭能力的变化。3.15只裸鼠随机分为3组,每组5只,分别接种转染siRNA-CD97细胞(抑制组),接种转染阴性对照siRNA-CD97细胞(阴性对照组),接种未转染细胞(正常对照组)。接种4周后,观察各组成瘤情况,比较成瘤率。结果:1.CD97-siRNA转染48小时后,RT-PCR方法检测结果表明CD97在mRNA水平表达下降70.12%;72小时后,Western blot方法检测结果表明CD97在蛋白水平的表达下降66.35%。2.CD97-siRNA转染72小时后,MTT法检测结果显示,与未转染组相比,细胞增殖率下降52.45%;流式细胞术检测结果显示,G1期细胞数量在未转染组、阴性对照组和CD97-siRNA转染组分别是14.48±3.45%、15.33±2.13%和39.79±3.05%,S期细胞数量分别是64.17±3.36%、60.35±5.21%和28.56±1.33%,出现G1→S期阻滞;同时,细胞凋亡率在未转染组、阴性对照组和CD97-siRNA转染组分别是7.24±0.35%、6.88±0.58和28.18±0.39%。3.CD97-siRNA转染72小时后,迁移和侵袭实验结果显示CD97-siRNA转染组迁移和侵袭细胞相对于未转染组分别下降了62.54±11.32%和67.13±14.03%。4.正常对照组和阴性对照组所有裸鼠均有瘤体形成,平均瘤重分别为5.255±0.571克和5.218±0.547克。CD97-siRNA转染组瘤体平均瘤重2.125±0.232克,与正常对照组相比有显著性差异(p<0.05),抑瘤率为58.1%。结论:1.CD97蛋白的表达强弱可以作为临床上判断胃癌细胞增殖、转移和侵袭能力的标志物之一,该分子可能成为抑制胃癌复发和转移的重要靶点;2.RNAi技术可以用于抑制CD97基因的表达;3.CD97-siRNA转染有望用于胃癌的基因治疗。第二章CD97-siRNA转染对胃癌细胞株MKN45生物学特性影响的分子机制的初步研究目的:研究CD97-siRNA转染诱导MKN45细胞G1期阻滞、细胞凋亡和抑制细胞体外迁移、侵袭能力的分子机制。方法:1.Western blot法检测CD97-siRNA转染后对胃癌细胞内细胞周期调节蛋白CyclinD1、CDK4和p27表达的影响。2.Western blot法检测CD97-siRNA转染后对胃癌细胞内凋亡调节因子Caspase家族表达的影响。3.Western blot法检测CD97-siRNA转染后对胃癌细胞内凋亡因子Bid、Bad、Bcl-2和Bcl-X_L表达的影响。4.Western blot法检测CD97-siRNA转染后对胃癌细胞MMP-7和TIMP-1表达的影响。5.Western blot法检测CD97-siRNA转染后对胃癌细胞PI3K/AKT表达的影响。结果:1.Western blot法检测结果表明:与未转染组和阴性对照组相比,CD97-siRNA转染72h后胃癌细胞Cyclin D1、CDK4的表达明显下调,而p27的表达明显上调。2.Westernblot法检测结果表明:与未转染组和阴性对照组相比,CD97-siRNA转染72h后胃癌细胞procaspase-9、-3的表达水平明显下降,cleaved-caspase-3的表达明显上升,而procaspase-8的表达无明显变化。CD97-siRNA是通过内源性途径诱导凋亡的。3.Western blot法检测结果表明:与未转染组和阴性对照组相比,CD97-siRNA转染72h后胃癌细胞Bcl-2的表达显著性下调,Bad的表达明显上调,Bcl-xl和Bid的表达无明显变化。4.Western blot法检测结果表明:与未转染组和阴性对照组相比,CD97-siRNA转染72h后胃癌细胞TIMP-1的表达明显上调,而MMP-7的表达明显下调。5.Western blot法检测结果表明:与未转染组和阴性对照组相比,CD97-siRNA转染72 h后胃癌细胞PI3K和AKT的磷酸化水平降低,这表明CD97分子可能是通过PI3K/AKT信号通路发挥作用。结论:1.CD97受体可能是通过PI3K/AKT信号通路发挥作用的。2.CD97-siRNA转染下调CD97分子的表达,PI3K及AKT的活性降低,进而上调p27、Bad、TIMP-1蛋白的表达,下调cyclin D1、CDK4、Bcl-2、MMP-7的表达,通过内源性凋亡途径活化caspase-3,从而抑制胃癌细胞的增殖、促进细胞凋亡并抑制肿瘤细胞的迁移、侵袭。更多还原

【Abstract】 INTRODUCTIONStomach cancer remained the fourth most common malignancy in the world in 2007,with one million new cases.Nearly 70%of new cases occured in developing countries.Generally,stomach cancer rates are about twice as high in men as in women. About 800,000 people worldwide died from stomach cancer in 2007.It ranks first among all causes of death from cancer in China,with an annual mortality rate of approximately 25.2 per 100.000.Cancer of the stomach is difficult to cure unless it is found in an early stage.The main treatments for stomach cancer are surgery,chemotherapy,and radiation therapy. Often the best approach uses two or more of these treatment methods.Detection of two or more than two tumor markers together will increase the rate of early diagnosis of stomach cancer.At present,CEA and CA19-9 are the two main tumor markers of stomach cancer.So,discovery of novel tumor marker is one of the research trend of tumor.Despite the recently reduced mortality rates due to both earlier detection and improved therapy,gastric cancer is still the second leading cause of cancer-related death worldwide.The development of new treatments is therefore crucial for improving the survival rates of gastric cancer patients.In the last few years,dedifferentiation markers and apoptotic factors have attracted considerable attention as possible targets for the diagnosis and therapy of cancer.CD97 is a member of a subfamily of classⅡG-protein-coupled receptors referred to as epidermal growth factor seven-transmembrane(EGF-TM7).CD97 protein possesses a unique hybrid structure that consists of varying numbers of N-terminal EGF-like domains coupled to a seven-span TM7 domain by a mucin-like spacer.In normal tissues,abundant expression of CD97 is only detected in macrophages and dendritic cells except for microglial cells,and in some T and B cells.To date,CD97 has been found abundant expression in several epithelial cancers,such as thyroid, gastrointestinal,pancreatic ductal and oral squamous cell carcinoma.Various evidence shows that CD97 plays an important role in tumor dedifferentiation,migration, invasiveness and metastasis.However,the role of CD97 on gastric carcinoma cell growth and the underlying mechanism remain to be elucidated.Part 1 Effects of CD97-siRNA transfection on biological characteristics in gastric carcinoma cell line MKN45Objective:To investigate the effect of CD97 gene silencing by small interfering RNA on proliferation,apoptosis,migration and invasion of gastric carcinoma cell line MKN45 in vivo and in vitro.Methods:1.Gastric carcinoma cell line MKN45 was adopted,the pairs of CD97-siRNA were designed according to the CD97 gene sequence and synthesized chemically.2.Forty-eight hours after CD97-siRNA transfection,the expression of CD97 at mRNA level was detected with RT-PCR.72 hrs after transfection,the expression of CD97 at protein level was detected with Western blot and Immunocytochemistry assays,MTT assay and FCM were used to evaluate the change of proliferation and apoptosis,and Transwell assay was used to evaluate the change of ability of migration and invasion in gastric carcinoma cells.3.15 nude mice were divided into 3 groups averagely.Group normal control: subcutaneous implant of gastric carcinoma cells with normal gastric carcinoma cells. Group negative control:subcutaneous implant of gastric carcinoma cells with nonsilencing RNA.Group inhibition:subcutaneous implant of gastric carcinoma cells with CD97-siRNA transfection.4 weeks after implantation,the morphology, histopathology and tumor inhibition rate were evaluated on every group.Results:1.Forty-eight hours after CD97-siRNA transfection,RT-PCR revealed that CD97 expression decreased 70.12%at mRNA level,and 72 h after CD97-siRNA transfection, Western blot revealed that CD97 expression decreased 66.35%at protein level.2.Seventy-two hours after CD97-siRNA transfection,MTT revealed that the rate of cell proliferation decreased 52.45%compared to the normal control group,and FCM revealed that the percentage of G1 stage cells of normal control,negative control and transfection group were 14.48±3.45%,15.33±2.13%and 39.79±3.05%respectively;the percentage of S stage cells were 64.17±3.36%,60.35±5.21%and 28.56±1.33% respectively.These data showed that CD97-siRNA restrained the conversion of G1→S stage.FCM revealed that the percentage of apoptotic cells of normal control,negative control and transfection group were 7.24±0.35%,6.88±0.58 and 28.18±0.39% respectively.3.Seventy-two hours after CD97-siRNA transfection,Transwell assay revealed that the number of migration and invasion cells decreased 62.54±11.32%and 67.13±14.03% respectively.4.Tumor formation can be found in every nude mice.Tumor tissue mean weights of the normal control and negative control group were 5.255±0.571g and 5.218±0.547g respectively.However,tumor tissue weight of CD97-siRNA transfection group was 2.125±0.232g.Tumor growth was inhibited significantly(p<0.05) and the inhibition rate was 58.1%.Conclusion:1.The status of CD97 expression can be used as a marker to evaluate the ability of proliferation,migration and invasion of gastric cancer,and this molecule can be used as an important target to suppress the recurrence and metastasis of gastric cancer.2.RNAi technique can be used to suppress the expression of CD97 gene.3.CD97-siRNA may be used as gene therapy means for gastric cancer.Part 2 A pilot study of the underlying molecular mechanism of CD97-siRNA transfection-induced biological characteristics in gastric carcinoma cell line MKN45Objective:To study the underlying molecular mechanism of CD97-siRNA-induced cell cycle arrest, apoptosis,and suppression of migration and invasion of gastric carcinoma cell line MKN45 in vitro.Methods:1.Western blot assay was used to determine the change of level of protein of Cyclin D1、CDK4 and p27 after CD97-siRNA transfection.2.Western blot assay was used to determine the change of level of protein of caspase family after CD97-siRNA transfection.3.Western blot assay was used to determine the change of level of protein of Bid、Bad、Bcl-2 and Bcl-X_L after CD97-siRNA transfection.4.Western blot assay was used to determine the change of level of protein of MMP-7 and TIMP-1 after CD97-siRNA transfection.5.Western blot assay was used to determine the effect of CD97-siRNA transfection on PI3K/AKT signal pathway.Results:1.Western blot assay revealed that CD97-siRNA transfection down-regulated the level of expression of Cyclin D1 and CDK4,and up-regulated the level of expression of p27 in MKN45 cells significantly.2.Western blot assay revealed that CD97-siRNA transfection down-regulated the level of expression of procaspase-9,-3,and up-regulated the level of expression of cleaved-caspase-3 significantly,however,had no significant influence on the level of expression of procaspase-8 in MKN45 cells.These data showed that CD97-siRNA triggers apoptosis of gastric cancer cells largely through the intrinsic pathway.3.Western blot assay revealed that CD97-siRNA transfection down-regulated the level of expression of Bcl-2 and up-regulated the level of expression of Bad significantly, however,had no significant influence on the level of expression of Bcl-x1 and Bid in MKN45 cells.4.Western blot assay revealed that CD97-siRNA transfection up-regulated the level of expression of TIMP-1,down-regulated the level of expression of MMP-7 in MKN45 cells significantly.5.Western blot assay revealed that CD97-siRNA transfection decreased the amount of phosphorylated PI3K and AKT in MKN45 cells significantly.These data showed that CD97 enhance the cancer growth probably through PI3K/AKT signal transduction pathway.Conclusion:1.The CD97 receptor enhance the cancer growth probably through PI3K/AKT signal transduction pathway.2.CD97-siRNA transfection may down-regulate the level of expression of CD97 gene, inactivate the PI3K and AKT,up-regulate the level of expression of p27, cleaved-caspase-3,Bad and TIMP-1,and down-regulate the level of expression of cyclin D1,CDK4,procaspase-9,-3,Bcl-2 and MMP-7,furtherly result in cell cycle arrest,apoptosis,and suppression of migration and invasion of gastric carcinoma cells.更多还原