Kiss-1基因启动子区的高甲基化在胃癌中的临床意义

发布时间：2018-08-23 10:33

【摘要】：目的:胃癌在我国是常见的消化道恶性肿瘤,其发病率和死亡率一直位于男女恶性肿瘤的前列,严重威胁着国民健康。胃癌的早期症状不明显,而且缺乏有效的监测指标,到医院就诊的患者往往已经达胃癌的中晚期,治疗花费大、疗效差、生存率低。因此,临床迫切需要寻找有效的肿瘤标记物和肿瘤治疗靶点,早期发现、早期诊断和早期治疗胃癌,阻断肿瘤进展、精准治疗和提高患者的预后。随着对肿瘤生物学行为的深入了解,越来越多证据表明,抑癌基因启动子区的异常甲基化在胃癌的发生和发展中扮演着重要的角色。对抑癌基因启动子区甲基化进行分析可以作为肿瘤早期监测和预防的手段。因此,本研究选取抑癌基因Kiss-1作为研究指标,采用甲基化特异性聚合酶链反应(MSP)方法检测胃癌组织和癌旁胃正常黏膜组织中Kiss-1基因启动子区的甲基化状态,探讨其在胃癌发生发展中的作用以及与临床病理资料之间的关系。方法:1研究对象及分组2014年9月至2016年6月期间,在河北医科大学第一医院行手术治疗的40例胃癌患者(术前均未进行放疗、化疗),其中男性32例,女性8例,平均年龄65.25±9.47岁。分组情况:胃癌组40例(均来自手术切除的肿瘤组织);对照组30例(选取前述患者癌旁胃正常黏膜组织,距肿瘤边缘5cm以上)。新鲜组织标本离体后立即置于入冰盒中,随即转入-80℃冰箱贮存待用。具体病例资料:临床TNM分期:Ⅰ+Ⅱ期15例、Ⅲ+IV期25例;肿瘤分化程度:中高分化26例、低分化14例;肿瘤浸润情况:侵透浆膜19例、未侵透浆膜21例;肿瘤病理组织学类型均为腺癌,其中伴有淋巴结转移24例,不伴淋巴结转移16例。所有癌组织及癌旁组织均经过术后病理检查确诊。2实验原理甲基化特异性pcr(msp)法基本原理:用亚硫酸氢钠处理基因组dna,未被甲基化的胞嘧啶将发生氧化性脱氨基作用变成尿嘧啶,而甲基化的胞嘧啶则不会发生这种变化,然后分别用甲基化引物和非甲基化引物对所测基因的同一段核苷酸序列进行扩增,扩增产物用琼脂糖凝胶电泳检测,凝胶成像系统扫描分析结果。3结果判定3.1如果只有甲基化引物扩增出相应的条带,判定kiss-1基因启动子区完全甲基化;3.2如果只有非甲基化引物扩增出相应的条带,判定kiss-1基因启动子区没有发生甲基化;3.3如果两种引物均能扩增出相应的条带,判定kiss-1基因启动子区不全甲基化。4统计学方法应用spss21.0统计软件分析所得数据,组间率的比较用?2检验,以p0.05为差异有统计学意义。结果:1kiss-1基因启动子区甲基化状况kiss-1基因启动子区甲基化阳性率在胃癌组为72.5%(29/40),在癌旁正常组为33.3%(10/30),组间比较差异具有统计学意义(p0.05)。2kiss-1基因启动子区甲基化状态与胃癌患者临床病理资料之间的关系胃癌组中:kiss-1基因启动子区甲基化阳性率在淋巴结转移组及tnm分期iii+iv组分别明显高于无淋巴结转移组及tnm分期i+ii组,与淋巴结转移、tnm分期相关(p0.05),与患者的性别、年龄和胃癌的病理分化程度、浸润情况无关(p0.05)。结论:1胃癌组中kiss-1基因启动子区甲基化显著高于癌旁正常组,提示kiss-1基因启动子区高甲基化可能参与了胃癌的发病过程。2kiss-1基因启动子区高甲基化多见于胃癌伴有淋巴结转移者和临床分期晚者,提示检测kiss-1基因启动子区甲基化状态有助于判断胃癌患者预后。3 Kiss-1基因启动子区CpG岛有潜力成为干预胃癌发生与进展的分子靶点。
[Abstract]:Objective: Gastric cancer is a common malignant tumor of digestive tract in China. The morbidity and mortality of gastric cancer are always in the forefront of malignant tumors of men and women, which seriously threaten the national health. Therefore, there is an urgent need to find effective tumor markers and therapeutic targets, early detection, early diagnosis and treatment of gastric cancer, blocking tumor progression, accurate treatment and improving the prognosis of patients. Methylation plays an important role in the occurrence and development of gastric cancer. Methylation analysis of promoter region of tumor suppressor gene can be used as a means of early detection and prevention of cancer. Methylation status of Kiss-1 gene promoter region in normal paragastric mucosa and its role in the development of gastric cancer and its relationship with clinicopathological data were investigated. Radiotherapy and chemotherapy were performed in 32 males and 8 females, with an average age of 65.25 (+ 9.47 years). The patients were divided into two groups: gastric cancer group (40 cases) and control group (30 cases) with normal gastric mucosa adjacent to the cancer, more than 5 cm from the edge of the tumor). Specific case data: clinical TNM staging: stage I+II 15 cases, stage III+IV 25 cases; tumor differentiation degree: 26 cases of high differentiation, 14 cases of poor differentiation; tumor infiltration: penetrating serosa 19 cases, not penetrating serosa 21 cases; tumor pathological types are adenocarcinoma, including 24 cases with lymph node metastasis, 16 cases without lymph node metastasis. All cancer tissues and adjacent tissues were confirmed by postoperative pathological examination. 2 Experimental Principle Methylation Specific PCR (msp) Basic Principle: Treating genomic DNA with sodium bisulfite, unmethylated cytosine converts oxidative deamination into uracil, whereas methylated cytosine does not undergo this change, and then uses them separately Methylation primers and non-methylation primers were used to amplify the same segment of the nucleotide sequence of the tested gene. The amplified products were detected by agarose gel electrophoresis. The results of gel imaging system scanning analysis showed that 3.1 if only methylation primers amplified the corresponding bands, the KiSS-1 gene promoter region was completely methylated; 3.2 if only methylation primers amplified the corresponding bands. Non-methylation primers amplified the corresponding bands, determined that KiSS-1 gene promoter region did not methylate; 3.3 If the two primers can amplify the corresponding bands, determine KiSS-1 gene promoter region incomplete methylation. Results: The positive rate of methylation in promoter region of KiSS-1 gene was 72.5% (29/40) in gastric cancer group and 33.3% (10/30) in adjacent normal group. The difference between the two groups was statistically significant (p0.05). The methylation status of promoter region of KiSS-1 gene was significantly different from that of gastric cancer patients. The positive rates of KiSS-1 promoter methylation in lymph node metastasis group and TNM stage I I I + IV group were significantly higher than those in non-lymph node metastasis group and TNM stage I + I I group, and were correlated with lymph node metastasis and TNM stage (p0.05). There was no correlation between KiSS-1 promoter methylation and gender, age, pathological differentiation and invasion of gastric cancer (p0.05). The hypermethylation of the promoter region of KiSS-1 gene in gastric cancer group was significantly higher than that in the adjacent normal group, suggesting that hypermethylation of the promoter region of KiSS-1 gene may be involved in the pathogenesis of gastric cancer. CpG island in the promoter region of Kiss-1 gene may be a potential molecular target for intervention in the occurrence and progression of gastric cancer.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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