【作者】 谢媛媛；

【导师】 李建生；

【作者基本信息】 郑州大学 ， 内科学， 2010， 硕士

【摘要】 大肠癌(colorectal cancer, CRC)包括结肠癌和直肠癌,是消化系统常见的恶性肿瘤。它的发病率在全世界恶性肿瘤的发病率中占第三位,在西方发达国家恶性肿瘤的发病率中占第二位。在我国,随着近年来生活水平的提高和饮食结构的改变,大肠癌的发病率也在日渐增高,已跃居我国恶性肿瘤发病率的第3-5位,并且,据预测我国大肠癌的发病率和死亡率在今后仍将逐渐上升。大肠癌的治疗效果并不理想,而且目前尚难实施有效地病因预防。但是,因为大肠癌具有比较明显的癌前病变及早期病变,且时间长达十余年,这为其筛查及早期诊断、早期治疗提供可能。近年来的研究已阐明大肠癌的发生发展是一个多步骤、多因素、多种基因改变协同作用的过程。在这些过程中,调控细胞基因表达的程序经常被改变。其中表观遗传修饰是基因改变的重要方式之一。表观遗传(epigenetics)是指不涉及基因组DNA序列改变而在细胞分裂过程中可以遗传给子代细胞的基因组修饰作用。DNA甲基化和组蛋白乙酰化修饰是表观遗传修饰的主要方式。由于DNA甲基化改变常常是恶性肿瘤发生发展过程中的早期事件,在大肠癌患者脱落细胞DNA检测中容易被发现,因此检测DNA甲基化状态改变也许可以作为一项新的大肠癌诊断方法,从而用于大肠癌的筛查和早期诊断。组织因子途径抑制物2(tissue factor pathway inhibitor 2, TFPI2)基因编码产物是广谱丝氨酸蛋白酶抑制物,广泛分布于人类肝、胰腺、肾、心、骨骼肌和前列腺等正常组织,体外可强烈抑制纤溶酶、胰蛋白酶、基质金属蛋白酶1(matrix metalloproteinases 1, MMP-1)和3(MMP-3)的活化,参与细胞外基质(extracellular matrix, ECM)的重塑过程,维持ECM的结构完整,调控肿瘤细胞的侵袭转移,在肿瘤细胞侵袭转移等一系列生理和病理过程中扮演重要角色。当TFPI2基因启动子区出现甲基化时,可从转录水平下调TFPI2基因表达,从而降低其调控肿瘤细胞侵袭转移的能力,促进恶性肿瘤的发生发展。目的本文应用甲基化特异性PCR (methylation-specific PCR,MSP)技术分析了30例大肠癌患者及30例正常对照者粪便中的TFPI2基因甲基化状态,并对同一份粪便标本用免疫化学法进行粪便隐血试验,用于比较,评估检测TFPI2基因甲基化状态改变的方法作为大肠癌无创性诊断方法的可行性。材料与方法选取2009年5月至2009年9月郑州大学第一附属医院收治的30例大肠癌患者为研究对象,其均经手术或内镜病理证实。30例大肠癌患者中,男17例,女13例,男女比例为1.3：1,年龄范围为21-82岁,平均年龄57岁。另选择30例年龄匹配的健康人为正常对照组,全结肠镜检查均显示全结肠粘膜未发现异常。正常对照组中男15例,女15例,男女比例为1：1,年龄范围为32-79岁,平均年龄为54岁。粪便标本于术前或内镜检查前收集,收集后立即送至实验室,置-70℃冰箱保存。所有研究对象入院或门诊检查时均用免疫化学法检测粪便隐血。对标本进行预处理后,用QIAamp DNA Stool Mini kit(QIAGEN,德国)试剂盒提取DNA并保存-20℃冰箱中。再按照The MethylampTM One-Step DNA Modification Kit (EPIGENTEK,美国)试剂盒操作说明书,进行DNA的亚硫酸氢盐修饰和纯化,置-70℃冰箱保存备用。以重亚硫酸氢盐修饰后的DNA作为甲基化特异性PCR的模板,检测TFPI2基因甲基化状态。实验结果采用SPSS17.0统计软件处理,基因频率采用直接计数法,各组间率的比较采用X2检验。检验结果以P<0.05有统计学意义。结果1在30例大肠癌患者粪便标本中,有19例(63.3%)标本检测出TFPI2基因启动子区甲基化,而30例正常对照者的粪便标本则无1例检测出TFPI2基因启动子区甲基化。2 30例大肠癌患者中,11例粪便隐血试验结果阳性(36.7%),并且11例隐血阳性结果的大肠癌患者粪便的TFPI2基因甲基化检测结果均为阳性。TFPI2甲基化检测诊断大肠癌的检出率(63.3%)与粪便隐血试验诊断大肠癌的检出率(36.7%)相比较,X2=4.267,P<0.05。3TFPI2基因甲基化状态改变与患者性别、年龄和肿瘤发生部位没有明显的相关性(P值均>0.05)。4TFPI2基因启动子区甲基化在Dukes分期A、B和C、D期中发生率分别为47.1%和84.6%,差异显著(P<0.05)；其次,低分化肿瘤组织中TFPI2基因启动子甲基化发生率为100%,中、高分化肿瘤组织中TFPI2基因启动子甲基化发生率为47.6%,二者差异非常显著(P<0.05)。结论1TFPI2基因甲基化是大肠癌患者粪便脱落细胞中常见的表观遗传学改变,并提示TFPI2作为一种抑癌基因,可能成为非侵入性大肠癌诊断方法的分子标志物之一。2TFPI2基因甲基化的阳性状态与大肠癌患者的年龄、性别和肿瘤部位无明显相关。3 TFPI2基因甲基化的阳性状态与大肠癌患者的肿瘤Dukes分期和组织分化程度有明显相关性。更多还原

【Abstract】 Colorectal cancer, including colon and rectal cancer, is a common digestive system cancer. The incidence of the cancer accounted for the third place in the world, and the second place in Western developed countries.In China, with the improvement of living standards and dietary changes in the structure in recent years, the incidence of colorectal cancer is rising gradually, which has leapt to the 3rd-5th place. And, according to forecasts, China’s colorectal cancer incidence and mortality rate will increase in the future. Treatments of colorectal cancer were not satisfactory, and the prevention is hard to implement effectively at present. In addition, screening,early diagnosis and early treatment are possible, because colorectal cancer has more obvious precancerous lesions and early lesions, which last more than ten years.In recent years, studies have clarified that the occurrence of colorectal cancer is a multi-step,multi-factor and a variety of genetically modified synergistic process. The programs of gene expression are often changed. Studies have shown that epigenetic is one of important ways to change modification of gene expression. Epigenetic is a modification of the genome, which dose not changes the genomic DNA sequence but can be transmitted to progeny cells in the process of cell division. DNA methylation and histone acetylating modification are the main forms of epigenetic modification. DNA methylation is often the early phase in the development of tumor, and can easily be detected in the stool of patients. Therefore, the detection of colorectal cancer-related genes methylation status in stool may become a new way of colorectal cancer screening and early non-invasive diagnosis.The product of TFPI2 gene encodes is a broad-spectrum serine protease inhibitor, which is widely distributed in human’s normal tissues such as liver, pancreas, kidney, heart, skeletal muscle and prostate, can strongly inhibit plasmin, trypsin, matrix metalloproteinase’s 1(matrix metalloproteinase’s, MMP-1) and 3 (MMP-3) activation in exterior, is involved in extracellular matrix (extracellular matrix, ECM) remodeling process to maintain the structural integrity of ECM and then to regulate of tumor cell invasion and metastasis, and plays an important role in a series of physiological and pathological processes, such as the invasion and metastasis of tumor cells. When the TFPI2 gene appears methylation, it can fall gene expression from the transcriptional level, thereby reducing its regulation ability of tumor cell invasion and metastasis and promoting the occurrence and development of malignant tumors.ObjectiveIn this paper, methylating-specific PCR(MSP) technique was used to analyses 30 cases of colorectal cancer patients and 30 normal controls stool TFPI2 gene methylation status, and stool occult blood test was done for the same stool samples with immunochemical method, which was used as a comparison to evaluate the feasibility of TFPI2 gene methylation in colorectal cancer as a non-invasive diagnosis method.Materials and methods60 cases including 30 colorectal cancers and 30 normal controls were randomly selected from the patients, who admitted to the first affiliated hospital of Zhengzhou University to take colonoscopy from May to September,2009.30 colorectal cancer patients,17 male and 13 female, male to female ratio of 1.3:1, age between 21 and 82 with an average age of 57. As for 30 cases of normal controls including 15 males and 15 females, male to female ratio of 1:1, age range 32 to 79 years, with an average age of 54 years, all colonoscopy were negative. Stool specimens were collected before colonoscopy or surgery from the 60 patients, then sent to the laboratory immediately and preserved in -70℃refrigerator. All patients admitted to hospital or clinic examination were detected by immunochemical stool occult blood. After pretreatment of specimens, DNA was extracted with QIAamp DNA Stool mini kit(QIAGEN,Germany) and saved in -20℃refrigerator. Then DNA was modified by Bisulfite and purified according to The MethylampTM One-Step DNA Modification Kit (EPIGENTEK, USA) operation instructions and saved in -70℃refrigerator. The methylation status of genes TFPI2 was detected with the DNA with bisulfate modification as the template of MSP. The results of the experiment were processed by statistical software SPSS 17.0, with gene frequencies obtained through direct counting method and each group rate compared by the X2 test. The test results signify statistically with P<0.05.Results1 Among 30 stool specimens of colorectal cancer patients,19 specimens (63.3%) were detected to show TFPI2 gene methylation. Meanwhile, no one detected TFPI2 gene methylaion among 30 normal controls.2 Among 30 stool specimens of colorectal cancer patients,11 patients with stool occult blood positive (36.7%), the results of TFPI2 gene methylaion of these patients were positive too. With TFPI2 methylation positive rate (63.3%) and stool occult blood testing positive rate (36.7%) compared, X2=4.267, P<0.05.3 TFPI2 gene methylation and patients sex, age and tumor location have no signification correlation (P>0.05).4 The positive rates of TFPI2 gene methylation in Dukes stages A,B and C,D,47.1% and 84.6% respectively, were significantly different(p<0.05). Secondly, poorly differentiated tumor tissue TFPI2 gene methylation positive rate was 100%. In well-differentiated tumor tissue TFPI2 gene methylation positive rate was 47.6%. The difference is significant (p<0.05).Conclusion 1 TFPI2 gene methylation is a common change in stool cells of colorectal cancer patients, and TFPI2, as a tumor suppressor gene, may be one of molecular markers which are used non-invasive diagnosis for colorectal cancer.2 TFPI2 gene methylation status arid colorectal cancer patient’s age, sex and tumor location have no signification correlation.3 The correlation between TFPI2 gene methylation status and colorectal cancer patient’s Dukes staging and tumor differentiation degree is obvious.更多还原