發(fā)布時(shí)間：2018-08-23 10:33

【摘要】：目的:胃癌在我國是常見的消化道惡性腫瘤,其發(fā)病率和死亡率一直位于男女惡性腫瘤的前列,嚴(yán)重威脅著國民健康。胃癌的早期癥狀不明顯,而且缺乏有效的監(jiān)測指標(biāo),到醫(yī)院就診的患者往往已經(jīng)達(dá)胃癌的中晚期,治療花費(fèi)大、療效差、生存率低。因此,臨床迫切需要尋找有效的腫瘤標(biāo)記物和腫瘤治療靶點(diǎn),早期發(fā)現(xiàn)、早期診斷和早期治療胃癌,阻斷腫瘤進(jìn)展、精準(zhǔn)治療和提高患者的預(yù)后。隨著對(duì)腫瘤生物學(xué)行為的深入了解,越來越多證據(jù)表明,抑癌基因啟動(dòng)子區(qū)的異常甲基化在胃癌的發(fā)生和發(fā)展中扮演著重要的角色。對(duì)抑癌基因啟動(dòng)子區(qū)甲基化進(jìn)行分析可以作為腫瘤早期監(jiān)測和預(yù)防的手段。因此,本研究選取抑癌基因Kiss-1作為研究指標(biāo),采用甲基化特異性聚合酶鏈反應(yīng)(MSP)方法檢測胃癌組織和癌旁胃正常黏膜組織中Kiss-1基因啟動(dòng)子區(qū)的甲基化狀態(tài),探討其在胃癌發(fā)生發(fā)展中的作用以及與臨床病理資料之間的關(guān)系。方法:1研究對(duì)象及分組2014年9月至2016年6月期間,在河北醫(yī)科大學(xué)第一醫(yī)院行手術(shù)治療的40例胃癌患者(術(shù)前均未進(jìn)行放療、化療),其中男性32例,女性8例,平均年齡65.25±9.47歲。分組情況:胃癌組40例(均來自手術(shù)切除的腫瘤組織);對(duì)照組30例(選取前述患者癌旁胃正常黏膜組織,距腫瘤邊緣5cm以上)。新鮮組織標(biāo)本離體后立即置于入冰盒中,隨即轉(zhuǎn)入-80℃冰箱貯存待用。具體病例資料:臨床TNM分期:Ⅰ+Ⅱ期15例、Ⅲ+IV期25例;腫瘤分化程度:中高分化26例、低分化14例;腫瘤浸潤情況:侵透漿膜19例、未侵透漿膜21例;腫瘤病理組織學(xué)類型均為腺癌,其中伴有淋巴結(jié)轉(zhuǎn)移24例,不伴淋巴結(jié)轉(zhuǎn)移16例。所有癌組織及癌旁組織均經(jīng)過術(shù)后病理檢查確診。2實(shí)驗(yàn)原理甲基化特異性pcr(msp)法基本原理:用亞硫酸氫鈉處理基因組dna,未被甲基化的胞嘧啶將發(fā)生氧化性脫氨基作用變成尿嘧啶,而甲基化的胞嘧啶則不會(huì)發(fā)生這種變化,然后分別用甲基化引物和非甲基化引物對(duì)所測基因的同一段核苷酸序列進(jìn)行擴(kuò)增,擴(kuò)增產(chǎn)物用瓊脂糖凝膠電泳檢測,凝膠成像系統(tǒng)掃描分析結(jié)果。3結(jié)果判定3.1如果只有甲基化引物擴(kuò)增出相應(yīng)的條帶,判定kiss-1基因啟動(dòng)子區(qū)完全甲基化;3.2如果只有非甲基化引物擴(kuò)增出相應(yīng)的條帶,判定kiss-1基因啟動(dòng)子區(qū)沒有發(fā)生甲基化;3.3如果兩種引物均能擴(kuò)增出相應(yīng)的條帶,判定kiss-1基因啟動(dòng)子區(qū)不全甲基化。4統(tǒng)計(jì)學(xué)方法應(yīng)用spss21.0統(tǒng)計(jì)軟件分析所得數(shù)據(jù),組間率的比較用?2檢驗(yàn),以p0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1kiss-1基因啟動(dòng)子區(qū)甲基化狀況kiss-1基因啟動(dòng)子區(qū)甲基化陽性率在胃癌組為72.5%(29/40),在癌旁正常組為33.3%(10/30),組間比較差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。2kiss-1基因啟動(dòng)子區(qū)甲基化狀態(tài)與胃癌患者臨床病理資料之間的關(guān)系胃癌組中:kiss-1基因啟動(dòng)子區(qū)甲基化陽性率在淋巴結(jié)轉(zhuǎn)移組及tnm分期iii+iv組分別明顯高于無淋巴結(jié)轉(zhuǎn)移組及tnm分期i+ii組,與淋巴結(jié)轉(zhuǎn)移、tnm分期相關(guān)(p0.05),與患者的性別、年齡和胃癌的病理分化程度、浸潤情況無關(guān)(p0.05)。結(jié)論:1胃癌組中kiss-1基因啟動(dòng)子區(qū)甲基化顯著高于癌旁正常組,提示kiss-1基因啟動(dòng)子區(qū)高甲基化可能參與了胃癌的發(fā)病過程。2kiss-1基因啟動(dòng)子區(qū)高甲基化多見于胃癌伴有淋巴結(jié)轉(zhuǎn)移者和臨床分期晚者,提示檢測kiss-1基因啟動(dòng)子區(qū)甲基化狀態(tài)有助于判斷胃癌患者預(yù)后。3 Kiss-1基因啟動(dòng)子區(qū)CpG島有潛力成為干預(yù)胃癌發(fā)生與進(jìn)展的分子靶點(diǎn)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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本文編號(hào)：2198811