【作者】 张开立；

【导师】 李宏；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的胃癌(Gastric Cancer)是我国最常见的、同时也是世界范围内发病率和死亡率均较高的消化道恶性肿瘤之一。由于起病隐蔽,临床病人多为晚期患者,死亡率高居全球恶性肿瘤死亡率的第二位。其发生具有多因素、多步骤、多基因和多途径的特点。目前对胃癌缺乏准确有效的早期诊断方法是造成胃癌高死亡率的主要原因。因此深入了解胃癌发生、发展及转移机制,制订预测、早期诊断和早期阻断的有效措施尤为重要。大连地区是胃癌高发区,具有比较特殊的地理地貌特点以及生态学环境。本地居民也有独特的生活饮食习惯和相对封闭的遗传学背景。而这些环境因素和遗传因素都在胃癌的发生过程中发挥作用,针对这些因素的流行病学调查将有助于我们进一步加深对胃癌发生的分子生物学机制的理解。研究大连地区胃癌发生发展过程中的分子改变及周围环境的影响有助于人们深入认识胃癌在该地区的发生机制,对于制定胃癌的预防,早期诊断和治疗策略具有重要的意义。已有的地理流行病学资料显示,大连常住居民有喜食海鲜烧烤及腌制食品的饮食习惯,而这些食物中存在大量亚硝酸盐类及多环芳烃类化合物等致癌物前体,其中很多化合物如苯并芘(benzo(a)pyrene,B[a]P)等可以作为致突变剂和致甲基化剂通过影响一系列遗传学、表观遗传学改变进而发挥致癌作用。环境中的化学致癌物多为间接致癌物,即致癌物前体。它们在细胞内经过代谢活化后才具有致癌活性。细胞色素P450(CYP450)就是催化此类代谢活化反应的一组氧化酶。它们分布于多种脏器主质细胞的滑面内质网上,主要负责包括环境致癌物在内的外源性化合物的代谢解毒与活化。某些活化产物能够损伤生物大分子或形成DNA加合物进而激活原癌基因导致癌症发生。研究表明,大多数已知的环境化学致癌物都是CYP450的作用底物。CYP1A1基因是P450超基因家族中的一个重要成员。它可以被多种内、外源性多环芳烃类化合物如苯并芘诱导表达。其基因产物为芳烃羟化酶,可将多环芳烃类化合物代谢活化为环氟类化合物而进一步发挥致癌、致突变作用。有关CYP1A1表达在多阶段胃癌发生中的作用少见报道。鉴于大连地区人群有进食含有过量致癌物前体食品的习惯,我们对本地区胃癌组织中CYP1A1的表达特点及其与多阶段胃癌发生的关系加以分析;同时对CYP1A1基因表达调控机制以及大连地区人群CYP1A1基因多态性分布进行了研究。我们在前期工作中发现了大连地区胃癌COX-2基因启动子区域异常甲基化导致的COX-2基因的表达缺失。这一现象虽然和以往的许多研究结果有所不同,但是我们认为其含义深远,即它强烈地提示我们大连地区胃癌的高发机制可能与其他地区胃癌的发生机制不同——主要依赖于一种异常甲基化的表观遗传学改变途径,这可能是大连地区胃癌发病率高的始因。而这种表观遗传学改变与大连地区特殊的环境因素及本地区人群遗传背景有关。表观遗传学改变是一种未涉及DNA序列变化的基因外调控机制,主要包括DNA甲基化、组蛋白修饰等。其中DNA甲基化是目前人们研究最为深入的一种表观遗传学修饰方式,主要发生在CpG二核苷酸序列的胞嘧啶上,并证实与多种肿瘤的发生密切相关。DNA甲基化的可诱导性和可逆性特点也为肿瘤发生机制探讨和肿瘤治疗提供了新的思路。结合大连地区地理流行病学特点以及生活饮食习惯,饮食中的许多成分都被证实是促甲基化剂。因此对本地区胃癌患者以及正常对照人群多基因多位点的DNA甲基化特点(也称CpG岛甲基化表型,CpG Island Methylator Phenotype,CIMP)比较分析就显得尤为有意义。不仅可以发现胃癌患者的CIMP型特征,而且可以判断未患个体的甲基化易感性,进而预测个体发病风险。为本地区胃癌的预防、预测和早期诊断提供理论指导。我们的研究目的在于通过检测特定区域肿瘤、癌前病变、癌旁相对正常以及非癌正常对照胃粘膜组织的遗传学和表观遗传学改变,判断遗传因素和环境因素在肿瘤发生过程中的作用及相互联系。探讨大连胃癌高发区胃癌发病机制,为胃癌临床早期诊断、预防乃至治疗方法的选择提供新的依据和思路。方法全部标本来自大连医科大学辽宁省(高校)癌症基因组重点实验室建立的人胃癌组织库。在冰冻组织微阵列技术和定点捕获技术基础上,应用免疫组化、RT-PCR、酶活性分析、Western蛋白印迹分析、PCR-SSCP、RFLP-PCR以及MS-PCR等方法,分别对不同病变胃粘膜组织和癌旁相对正常组织中CYP1A1基因表达特点进行分析,并对其基因表达调控机制如AhR(Aryl hydrocarbon receptor)基因共表达及核易位以及该基因MspⅠ和Exon7多态性与肿瘤易感性的关系进行探索;同时应用甲基化特异性PCR检测了5个常用CIMP鉴定位点(p16,hMLH1,MINT1,MINT2,MINT31)以及与胃癌的恶性表型密切相关的CDH1基因5’启动子区的甲基化状态。实验数据采用SPSS12.0软件包应用Kruskal-Wallis和Mamm Whitney检验方法进行统计分析。结果一.CYP1A1基因在不同病变胃粘膜组织中的表达情况免疫组化结果显示:在癌旁相对正常、癌前病变以及胃癌组中,CYP1A1基因表达率分别为11.1%(1/9),60%(12/20)和86.0%(37/43),组间差别显著,具有统计学意义(p<0.005)。RT-PCR和Western印迹杂交结果基本与之符合。二. AhR基因在不同病变胃组织中的表达与核易位特点在39例胃癌样本中,有37例AhR基因表达,同时这37例样本都发生了AhR蛋白的核内表达(核易位),总阳性率和核易位率均为94.9%(37/39);在6例癌旁相对正常胃粘膜组织标本中,有3例表现为弱阳性(+),阳性率为50%,其中只有1例发生核易位,核易位率仅为16.7%(1/6)。计数资料统计学检验结果显示:不同病变胃粘膜组织(胃癌和癌前病变)中的AhR基因表达水平要明显高于癌旁相对正常组织(p<0.05);AhR蛋白的核内表达(核易位)在与CYP1A1基因的高表达存在正相关(rs=0.437,p<0.01)。三.CYP1A1基因多态性分析1、MspI多态性分析在胃癌组中,m1m1、m1m2和m2m2基因型频率分别为43.3%、45%和11.7%;在正常对照组为45.6%、49.1%和5.3%。两组间基因频率差别无统计学意义,从而证明CYP1A1基因的Msp I多态性与胃癌易感性无显著相关性。2、Exon 7多态性分析胃癌组Ile/Ile、Ile/Val Val/Val基因型频率分别为Ile/Ile型26.7%、50%和23.3%;正常对照组为52.6%、28.1%和19.3%。同样,组间基因频率差别无统计学意义。3、CYP1A1基因多态性与表达的关系RT-PCR和免疫组化结果基本一致:易感基因型m2m2和Val/Val个体CYP1A1表达率均为100%。而非易感基因型个体CYP1A1表达率为54-72%。组间统计学有显著差异(p<0.05),说明CYP1A1基因多态性可以明显地影响酶的表达量及活性。四.大连地区进展期胃癌的CIMP分布特征CIMP-H在胃癌、癌前以及癌旁相对正常组织中的发生率分别为51.1%(24/47)、23.8%(5/21)和0%(0/16);CIMP-L分别为38.3%(18/47)、71.4%(15/21)和75%(12/16);而CIMP-N则分别为10.6%(5/47)、4.8%(1/21)和25%(4/16),在不同进展期胃癌组间差异显著(p=0.001)。结果说明多位点甲基化与胃癌进展有关。随着胃癌的演进,甲基化基因种类明显增多,甲基化程度增强。非癌患者检测结果全部31例正常对照中无CIMP-H型个体,但出现38.7%(12/31)的CIMP-L个体。除p16外的其他4个位点的甲基化情况与胃癌组相比存在较大差异,具有统计学意义(p=0-0.008)。五.CDH1启动子甲基化检测胃癌粘膜组织中CDH1基因启动子甲基化率为48.9%(23/47),与胃癌CIMP型、进展、年龄因素及肿瘤淋巴结转移密切相关。六.大连地区胃癌CIMP分布与CYP1A1基因表达的相关性分析CYP1A1在CIMP-H组中的检测阳性率为100% (20/20),在CIMP-L/N组中的检测阳性率为62.5% (10/16),组间差异显著(p<0.01)。结论一.CYP1A1基因表达是大连地区胃癌发病过程中的一个重要分子事件,出现于胃癌发生早期并贯穿始终,在不同进展期胃癌组织中CYP1A1表达水平不同。可以把它作为衡量环境化学致癌因子影响和判断大连高发区胃癌病变程度的潜在分子标记物;二.大连地区胃癌中CYP1A1基因的高表达是由外源环境化学因素诱导并由AhR介导途径实现;三.CYP1A1基因多态性与大连地区胃癌易患性未见明显相关性。但不同基因型个体CYP1A1表达及酶活性存在显著差异。这说明,易感基因型个体易被诱导表达且酶活性增高,但由于本地区可能存在较强有力的环境暴露因子,因而导致易感基因型及非易感基因型个体的易患性高度都可超过阈值而发病;四.胃癌不同病变组与正常对照组中CIMP型分布有显著差异,说明大连高发区胃癌的发生可能依赖于一种异常甲基化途径;异常甲基化在本地区胃癌发生过程中属早期事件,并随疾病的演进而增强;五.大连胃癌高发区人群本身就存在较大的甲基化压力或者CIMP易患性,这可能与环境中的某些致甲基化剂的Ⅰ相代谢活化有关。综上所述,大连地区存在较强的环境致变因素。可引起遗传学及表观遗传学的一系列改变而始动肿瘤的形成并促进演进。对于本地区人群采取积极有效的隔离阻断措施(如指导改变本地区常住居民的饮食习惯、去甲基化剂的应用等)以减少环境暴露因子的影响,对于干预本地区胃癌的发生、降低罹病风险和病死率等是迫切而且必要的。更多还原

【Abstract】 Backgrounds and Objectives:Gastric cancer (GC) is one of the commonest gastric-intestinal malignancies in China with higher morbidity and mortality in the world. Due to lack of early diagnosis, prevention and treatments, GC remains the second killer malignancy among clinical patients around the world. Its development is a multi-factor, multi-step process in which many genes and several signaling pathways are involved. Thus, it is very important to further study the molecular mechanism of GC occurrence, development and metastasis in order to establish effective measures of early diagnosis and treatment of GC.Dalian belongs to GC at-risk regions in China. Its special geographical and physiognomy characteristics (a coastal and mountainous area located at the far south of Northeast China), ecological environment, the particular diet habits of its native residents, and relatively close genetic backgrounds play key roles in the development of GC. So Epidemiologic studies about this area seem significant to further understand the molecular mechanism of GC development and take effective measures of early prevention, diagnosis and treatment of GC.Epidemiologic studies have demonstrated that people in this area were accustomed to eating toasted or salted seafood and meat. These foods are rich in gastrocarcinogens such as heterocyclic amines, polycyclic aromatic hydrocarbons, and the well-known DNA methylators such as nitrite and benzo(a)pyrene which may enhance the risk of genetic and epigenetic alterations.Many chemicals in the environment are indirect pro-carcinogens and can be catalyzed to activated forms before playing their carcinogenic roles in the target cells. Cytochrome P450 (CYP450) enzymes are a group of oxidases that mainly distribute in smooth endoplasmic reticulum of cells and can activate or inactivate many types of exogenous compounds. Some activated products can play their carcinogenic roles by impairing biomacromolecules and/or forming DNA adducts in the target cells. It has been recognized that most of the known environmental carcinogens are the substrate of CYP450 enzymes.CYP1A1 is a major member of CYP450 superfamily and can be induced by many kinds of endogenous or exogenous compounds such as benzo(a)pyrene. The product of CYP1A1 gene is a kind of aryl hydrocarbon hydroylase which involves in tumorigenesis by activating polycyclic aromatic hydrocarbons (PAHs). The studies so far performed on the role CYP1A1 expression in stepwise gastrocarcinogenesis is lesser known. Since the people in Dalian region are used to eating some food that contain an overdose of procarcinogens, the status of CYP1A1 expression in Dalian gastric cancers and its potential link with tumor formation were investigated. Meanwhile, the studies about the modulation mechanism of CYP1A1 expression and CYP1A1 polymorphism distribution in the population of Dalian area were performed.Our previous work demonstrated that abnormal methylation of the COX-2 promoter could result in infrequent expression of COX-2 in Dalian GCs, as was inconsistent with many viewpoints of that time. However, it strongly suggested us that the mechanism of Dalian GCs development might be different from that of other areas, which was very important. So we speculated that the development of Dalian GCs might depend on epigenetic modulation of DNA abnormal methylation which could be chief criminal of Dalian GCs. The epigenetic modulation could be closely related with special environmental factors and genetic backgrounds of Dalian residents.Epigenetic changes influence gene transcription without alterations in DNA sequence including DNA methylation and chemical modification of histone. DNA methylation is a major category of epigenetic change often occurring in the cytosine of CpG islands located in gene promoter regions, which is thought to be closely correlated with tumorigenesis. The inducibility and reversibility of DNA methylation provide us a new thought about tumor development and treatment. Since the Epidemiologic characteristics and the traditional diet habits (food containing many methylating agents) in this region , it is significantl to compare DNA methylation of multi-loci between normal population and Dalian GC patients (also called CpG island methylator phenotype, CIMP). This study not only can explore the CIMP profiling of Dalian GCs but also can estimate the GC risk of normal people by measuring their methylation susceptibility. Thus it can provide certain theory basis for prevention, early diagnosis and treatment of GCs in this region.Now our study aims at exploring the role of genetic and environmental factors in tumorigenesis and the relation between them through testing genetic and epigenetic changes of GC tissues, premalignant tissues, GC surrounding tissues and noncancerous mucosa, which can provide some new evidences and thoughts for early diagnosis, prevention, and treatment of clinical GCs.Materials and methods:All specimens in this study were selected from the Human Frozen Gastric Tissue Bank of the Cancer Institute, Liaoning Laboratory of Cancer Genomics, Dalian Medical University. By the methods of frozen embedded tissue array immunohisto- chemistry, RT-PCR, EROD, Western blotting, SSCP, RFLP-PCR and MS-PCR, the expression pattern of CYP1A1 in different gastric tissues, the modulation machinery of this gene expression (concurrent expression of AhR-Aryl hydrocarbon receptor and nuclear translocation) and the correlation between its MspⅠand Exon7 polymorphism and tumor susceptibility were explored. Meanwhile, a paralleled study about five CpG island methylator phenotype (CIMP)-associated genes (p16, hMLH1, MINT1, MINT2, and MINT31) and the methylation statuses of the 5′-promoter region of CDH1( it was closely related with malignant phenotype of GCs) were conducted by methylation- specific polymerase chain reaction (PCR). The data were statistically analyzed by Kruskal-Wallis and Mamm Whitney with SPSS 12.0 software.Result:1. The expression status of CYP1A1 gene in different gastric mucosa tissues.The results of IHC displayed that the rates of CYP1A1 expression were 11.1%(1/9)in GC surrounding tissues, 60%(12/20)in premalignant tissues and 86.0%(37/43)in GC tissues. The difference was significant between different groups (p<0.005). The results of RT-PCR and Western blotting were almost consistent with the above ones.2. The expression pattern and nuclear translocation of AhR in different gastric tissues.The positive staining of AhR was found in 94.9%(37/39)of GCs and 50% (3/6) of non-cancerous mucosa. Furthermore, the incidences of AhR nuclear translocation were 94.9% (37/39) in GCs and 16.67% (1/6) in non-cancerous mucosa. Statistical analysis showed that the incidences and levels of AhR expression increased significantly in abnormal gastric tissues (including GC and premalignant tissues) in comparison with that of non-cancerous ones (p<0.05) and AhR nuclear translocation was positively correlated with CYP1A1 up-regulation in GC tissues (rs=0.437,p<0.01).3. The analysis of CYP1A1 gene polymorphism.3.1 The analysis of MspI polymorphismThe frequencies of m1m1, m1m2 and m2m2 genotypes in GC group were 43.3%, 45% and 11.7% and those of control group were 45.6%, 49.1% and 5.3%, respectively. Statistical analyses revealed no significant difference of genotype frequencies between cancer and control groups. So MspI polymorphism of CYP1A1 gene was not significantly correlated with the susceptibility of GCs.3.2 The analysis of Exon 7 polymorphismThe rates of Ile/Ile, Ile/Val and Val/Val genotypes in GC group were 26.7%, 50% and 23.3% and those of control group were 52.6%, 28.1% and 19.3%, respectively. Statistical analyses revealed no significant difference of genotype frequencies between cancer and control groups.3.3 The relation between the polymorphism of CYP1A1 gene and its expression.The results of RT-PCR and IHC displayed that the expression rate of CYP1A1 in the population with genotypes m2m2 and Val/Val was100% while 54-72% in the population with genotypes m1m1 and Ile/Ile. Statistical analysis showed significant difference between the two group(p<0.05),which revealed that the polymorphism of CYP1A1 gene could greatly influence its expression level and enzyme activity.4. The distribution pattern of CIMP in evolutional Dalian GCs.The frequencies of CIMP-H were 51.1% (24/47) in GC group, 23.8% (5/21) in premalignant lesions, and 0% (0/16) in noncancerous mucosa. CIMP-L were 38.3% (18/47) in GCs, 71.4% (15/21) in premalignant, and 75% (12/16) in noncancerous tissues, respectively. CIMP-N were 10.6% (5/47) in GCs, 4.8% (1/21) in premalignant, and 25% (4/16) in noncancerous tissues. Significant statistical difference existed in different stages of GCs (p=0.001), which revealed that the methylation of gene multi-site was related with the development of GCs. With the evolution of GCs, the number and intensity of gene methylation increased. Among the 31 gastric biopsies obtained from GC-free patients, None exhibited CIMP-H, whereas 12/31 (38.7%) were found with CIMP-L. In comparison with the data obtained from the noncancerous mucosa of GC-bearing patients, the frequencies of hMLH1, MINT1, MINT2 and MINT31 methylation were significantly lower (p=0.000–0.008) except that of p16 methylation.5. The methylation status of CDH1 promoter.The methylation frequency of CDH1 promoter was 48.9%(23/47)in GC mucosa tissues, which was closely related with CIMP, development, age and lymph node metastasis of GCs.6. The correlation analysis between CIMP distribution and CYP1A1 expression in Dalian GCsThe expression frequencies of CYP1A1 were 100% (20/20) in CIMP-H group and 62.5% (10/16) in CIMP-L/N group. There was significant difference between the two groups(p<0.01).Conclusions:1. The expression of CYP1A1 gene is an important molecular event throughout the whole gastric tumorigenesis in Dalian area whose expression level varies with the different development stages of GCs. In this regard, expression of CYP1A1 may become a potential biomarker in exploring the influence of environmental chemical carcinogen on health and judging the degree of Dalian GCs;2. The high level of CYP1A1 expression is induced by exogenous chemicals through AhR pathway;3. No significant correlation is found between the polymorphism of CYP1A1gene and the susceptibility of Dalian GCs. The expression level and enzymatic activity of CYP1A1 vary in different genotype individuals. CYP1A1 level and enzymatic activity are relatively high in susceptible genotype individuals. However, because of strong environmental carcinogens in the region, regardless of susceptible and not susceptible genotype individuals, they may be susceptible to GC.4. The distribution of CIMP is greatly different between GC group and control group, which reveal the high risk of Dalian GCs consists in a kind of abnormal methylation pathway. The abnormal methylation is an early molecular event in the development of GCs and its intensity increase with the evolution of disease.5. The strong susceptibility of methylation and/or CIMP in GC high-risk Dalian population may be closely related with the first stage metabolic activity of certain environmental methylators. In summary, there are many environmental mutagenic agents in Dalian. They may bring about a series of genetic and epigenetic changes which are closely linked with tumor formation. Therefore, it is highly possible to reduce the exposure of the population in this area to environmental chemical factors by giving people some guidance (for example, change the diet habits of native residents or adopt demethylators), which is in urgent need and would be essential to interfere with GC formation and to decrease disease risk and mortality.更多还原