【摘要】：目的:通過(guò)對(duì)胃癌及癌旁組織基因表達(dá)情況的檢測(cè),分析ERCC1(人切除修復(fù)交叉互補(bǔ)基因1)基因啟動(dòng)子區(qū)甲基化的狀況及DNMT1(DNA甲基轉(zhuǎn)移酶1)基因的表達(dá)在胃癌演變過(guò)程中的作用。在消化系統(tǒng)惡性腫瘤中,胃癌是主要的致死疾病之一,大部分患者診斷時(shí)已經(jīng)為晚期,所以胃癌的早期診斷尤為重要。胃癌產(chǎn)生與演變是一個(gè)復(fù)雜的病變過(guò)程,它受到多因素的共同調(diào)控,其中癌基因的非正常激活與抑癌基因轉(zhuǎn)錄功能的失活是兩個(gè)極其重要的原因,表觀遺傳學(xué)層面上的改變?cè)诓∽冞^(guò)程中發(fā)揮著重要的功能,最普遍的表觀遺傳學(xué)改變就是DNA甲基化。方法:(1)青島市市立醫(yī)院2014年1月至2014年10月經(jīng)普外科切除的新鮮腫瘤標(biāo)本共60例,經(jīng)病理組織學(xué)診斷為為原發(fā)性胃癌,術(shù)前未行抗腫瘤治療,比如放療化療,擁有完整的臨床資料。癌組織取自癌病變區(qū)域的中心,并取距離癌組織至少5cm區(qū)域組織作為癌旁組織。分離收集到的標(biāo)本組織中的DNA,然后使用亞硫酸鈉修飾方法對(duì)所得的DNA進(jìn)行裝飾,采用聚合酶增鏈?zhǔn)椒磻?yīng)技術(shù)對(duì)上述DNA進(jìn)行擴(kuò)大增殖。使用MSP方法對(duì)胃癌組織及癌旁組織ERCC1基因甲基化情況進(jìn)行測(cè)驗(yàn),并檢驗(yàn)ERCC1和DNMT1表達(dá)水平。分析探討在腫瘤演變過(guò)程中兩個(gè)基因的所發(fā)揮的功效。(2)對(duì)收集到的60例標(biāo)本組織中的RNA進(jìn)行分離,然后進(jìn)行逆轉(zhuǎn)錄反應(yīng),形成單鏈DNA,接著應(yīng)用PCR技術(shù)擴(kuò)大增殖,用熒光定量逆轉(zhuǎn)錄多聚合酶鏈RT-PCR技術(shù)對(duì)基因ERCC1、DNMT1的m RNA表達(dá)水平進(jìn)行檢驗(yàn)。(3)用免疫組織化學(xué)方法檢測(cè)癌組織和癌旁組織中的ERCC1及DNMT1蛋白表達(dá)水平狀態(tài),探究它們?cè)谖赴┭葑冞^(guò)程中所發(fā)揮的功效。使用SPSS17.0軟件對(duì)試驗(yàn)得到的數(shù)據(jù)進(jìn)行解析,使用χ2檢驗(yàn)方法于計(jì)數(shù)資料之間的比較中,組間的比較t檢驗(yàn)進(jìn)行處理;以P0.05作為評(píng)判標(biāo)準(zhǔn),差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果:(1)應(yīng)用MSP反應(yīng)檢測(cè)出胃癌組織ERCC1啟動(dòng)子區(qū)甲基化率為68.3%明顯高于正常組織23.3%,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。(2)應(yīng)用RT-PCR反應(yīng)方法進(jìn)行檢測(cè),在ERCC1表達(dá)下調(diào)的胃癌組織中DNMT1的表達(dá)高于ERCC1表達(dá)正常的組織,表達(dá)水平差別有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)應(yīng)用免疫組織化學(xué)方法檢驗(yàn),ERCC1在癌旁組織中的表達(dá)陽(yáng)性率為93.3%,明顯高于胃癌組織56.7%,差異有統(tǒng)計(jì)學(xué)意義(χ2=21.21,P0.001)。DNMT1在胃癌組織中的表達(dá)陽(yáng)性率為71.7%,明顯高于癌旁組織16.7%,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。(4)胃癌組織中ERCC1基因啟動(dòng)子區(qū)甲基化狀態(tài)與ERCC1 m RNA表達(dá)之間的關(guān)系,在41例ERCC1 m RNA陰性表達(dá)的胃癌組織中,檢測(cè)出38例該基因啟動(dòng)子區(qū)甲基化,而在ERCC1 m RNA表達(dá)陽(yáng)性的19例胃癌組織中,僅檢測(cè)出3例基因啟動(dòng)子甲基化,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。(5)胃癌組織中ERCC1與DNMT1蛋白表達(dá)之間的相關(guān)性分析,胃癌組織中的ERCC1與DNMT1蛋白的表達(dá)呈負(fù)相關(guān)(P0.001).結(jié)論:(1)在胃癌組織中,可發(fā)現(xiàn)ERCC1基因啟動(dòng)子區(qū)域發(fā)生高甲基化改變,并且同時(shí)伴有該基因蛋白的表達(dá)減低,和胃癌的發(fā)生存在一定的關(guān)系。(2)胃癌組織中ERCC1基因的m RNA表達(dá)低于癌旁組織,m RNA的低表達(dá)與ERCC1基因啟動(dòng)子區(qū)Cp G島高甲基化有關(guān)。(3)胃癌組織中的ERCC1與DNMT1蛋白的表達(dá)呈負(fù)相關(guān),胃癌組織中的DNMT1的m RNA及蛋白表達(dá)水平明顯上升,此改變或與抑癌基因ERCCl發(fā)生高甲基化存在一定的聯(lián)系,造成ERCC1 m RNA表達(dá)減少或缺失,基因蛋白表達(dá)同樣降低。
[Abstract]:Objective: To analyze the promoter methylation of ERCC1 gene and the role of DNMT1 gene expression in the development of gastric cancer by detecting gene expression in gastric cancer and adjacent tissues. The generation and evolution of gastric cancer is a complicated pathological process, which is regulated by many factors. Abnormal activation of oncogenes and inactivation of transcriptional function of tumor suppressor genes are two extremely important reasons. Epigenetic changes are found in gastric cancer. DNA methylation is the most common epigenetic change that plays an important role in the pathogenesis of gastric cancer. Methods: (1) A total of 60 fresh tumor specimens from Qingdao Municipal Hospital from January 2014 to October 2014 were surgically removed and diagnosed as primary gastric cancer by histopathology. Preoperative anti-tumor treatment, such as radiotherapy and chemotherapy, was completed. Cancer tissues were taken from the center of the lesion area and taken at least 5 cm away from the cancer tissue as adjacent tissues. DNA was isolated from the collected tissues and then decorated with sodium sulfite. The DNA was amplified and proliferated by polymerase chain reaction (PCR). Methods The methylation of ERCC1 gene in gastric cancer and adjacent tissues was tested, and the expression levels of ERCC1 and DNMT1 were examined. The expression of ERCC1 and DNMT1 was detected by fluorescence quantitative reverse transcription polymerase chain reaction (FQRT-PCR). (3) The expression of ERCC1 and DNMT1 proteins in cancer tissues and adjacent tissues was detected by immunohistochemistry to explore their effects on the development of gastric cancer. Results: (1) The methylation rate of ERCC1 promoter region in gastric cancer tissues detected by MSP reaction was 68.3% higher than that in normal tissues by 23.3%. The difference was statistically significant (P 0.001). (2) The expression of DNMT1 in gastric cancer tissues with down-regulation of ERCC1 was significantly higher than that in normal tissues (P 0.05). (3) The positive rate of ERCC1 expression in adjacent tissues was 93.3% by immunohistochemistry. The positive rate of DNMT1 expression in gastric cancer was 71.7%, which was significantly higher than that in adjacent tissues (P 0.001). (4) The relationship between the methylation status of ERCC1 gene promoter region and the expression of ERCC1 m RNA in gastric cancer tissues was negative in 41 cases. Methylation of the promoter region was detected in 38 gastric cancer tissues, but only 3 of 19 gastric cancer tissues with positive ERCC1 m RNA expression were detected in 3 cases (P 0.001). (5) Correlation analysis between the expression of ERCC1 and DNMT1 protein in gastric cancer tissues, ERCC1 and DNMT1 eggs in gastric cancer tissues. Conclusion: (1) Hypermethylation of the promoter region of ERCC1 gene can be found in gastric cancer tissues, and the expression of ERCC1 gene protein is decreased, which is associated with the occurrence of gastric cancer. (2) The expression of ERCC1 gene in gastric cancer tissues is lower than that in adjacent tissues, and the expression of M RNA is lower than that in adjacent tissues. (3) The expression of DNMT1 protein was negatively correlated with ERCC1 in gastric cancer tissues. The expression of DNMT1 m RNA and protein was significantly increased in gastric cancer tissues. This change may be related to the hypermethylation of tumor suppressor gene ERCCl, resulting in the decrease or deletion of ERCC1 m RNA expression and gene protein surface. The same has been reduced.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.2

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本文編號(hào)：2198262