本文關(guān)鍵詞：胃癌相關(guān)性線粒體D-環(huán)多態(tài)位點(diǎn)與8-羥基脫氧鳥(niǎo)苷表達(dá)的關(guān)系　出處：《河北醫(yī)科大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 胃癌 8-OHdG D-Loop區(qū) 氧化損傷 臨床特征 發(fā)病風(fēng)險(xiǎn) 預(yù)后

【摘要】：目的:胃癌(gastric cancer,GC)在我國(guó)惡性腫瘤的死亡率中居首位,就診患者以中晚期為主,易轉(zhuǎn)移復(fù)發(fā),預(yù)后差,嚴(yán)重威脅了人們的生命健康,早期診斷是改善胃癌預(yù)后的有效方法之一。近年來(lái),DNA氧化性損傷與胃癌發(fā)病的關(guān)系成為了研究熱點(diǎn)。氧化損傷會(huì)對(duì)蛋白質(zhì)、脂類(lèi)及DNA等生物大分子的結(jié)構(gòu)和功能造成損傷,并由此導(dǎo)致基因突變、細(xì)胞癌變及個(gè)體衰老等現(xiàn)象。線粒體DNA(mitochondrial DNA,mt DNA)處于高氧環(huán)境中,損傷修復(fù)系統(tǒng)不完整,缺乏組蛋白的保護(hù),因此與核基因相比更易受到活性氧(reactive oxygen species,ROS)的損傷。D-環(huán)區(qū)(D-Loop區(qū))是mt DNA分子復(fù)制及轉(zhuǎn)錄的調(diào)控中心,此區(qū)域成為了mt DNA突變熱點(diǎn),我們前期研究已經(jīng)鑒定了和胃癌發(fā)病風(fēng)險(xiǎn)相關(guān)聯(lián)的線粒體D-環(huán)區(qū)多態(tài)位點(diǎn),73G、16519C及523-524Del基因型可能增加了胃癌的發(fā)病風(fēng)險(xiǎn),16362C及309C insert基因型則可能降低了胃癌的發(fā)病風(fēng)險(xiǎn),并推論這些位點(diǎn)可能改變了線粒體的氧化水平而引起胃癌。氧自由基攻擊DNA所形成的產(chǎn)物8-羥基脫氧鳥(niǎo)苷(8-hydroxy-2-Deoxyguanosine,8-OHd G),在DNA復(fù)制時(shí)可引起G:C-T:A顛換突變,可作為外源性及內(nèi)源性因素對(duì)DNA氧化損傷的生物學(xué)標(biāo)志物。目前在鼻咽癌、肺癌、肝癌、卵巢癌、宮頸癌等惡性腫瘤中,研究發(fā)現(xiàn)8-OHd G的表達(dá)水平明顯高于正常,可用于評(píng)估惡性腫瘤的進(jìn)展及預(yù)后。本研究通過(guò)免疫組織化學(xué)的方法,通過(guò)檢測(cè)胃癌組織中8-OHd G的表達(dá)水平以反應(yīng)氧化損傷的程度,分析氧化損傷與胃癌發(fā)病風(fēng)險(xiǎn)相關(guān)的線粒體DNA D-環(huán)區(qū)單核苷酸多態(tài)性(single nucleotide polymorphisms,SNPs)之間的關(guān)系,以探索胃癌的發(fā)生發(fā)展機(jī)制,并分析氧化損傷與胃癌預(yù)后及臨床特征的關(guān)系,為胃癌的早期診斷及預(yù)后監(jiān)測(cè)提供新思路。方法:1研究對(duì)象、標(biāo)本采集及隨訪:選取2007年3月-2008年8月于河北醫(yī)科大學(xué)第四醫(yī)院外三科行胃癌根治術(shù)的131例患者,術(shù)前均經(jīng)內(nèi)鏡檢查及組織學(xué)病理確診為胃癌。所有患者術(shù)前均未行化療和/或放療等臨床治療,術(shù)后病理結(jié)果均經(jīng)河北醫(yī)科大學(xué)第四醫(yī)院病理科兩名以上經(jīng)驗(yàn)豐富的醫(yī)師確診。詳細(xì)記錄所有患者的性別、年齡、腫瘤部位、腫瘤直徑及臨床分期等臨床特征,通過(guò)門(mén)診復(fù)查、信件或電話對(duì)胃癌患者術(shù)后生存狀況進(jìn)行隨訪,隨訪截止時(shí)間為2011年8月。術(shù)后胃癌組織的收集均經(jīng)過(guò)河北醫(yī)科大學(xué)第四醫(yī)院倫理委員會(huì)批準(zhǔn)。2線粒體DNA擴(kuò)增及測(cè)序:將已提取的鑒定合格后的DNA,采用PCR技術(shù)擴(kuò)增目的片段,上游引物為:5'-CCCCATGCTTACAAGCAA-GT-3',下游引物為:5'-GCTTTGAGGAGGTAAGCTAC-3'。擴(kuò)增產(chǎn)物經(jīng)瓊脂糖凝膠純化后,進(jìn)行測(cè)序,所用儀器為ABI PRISM#174;3730XL測(cè)序儀。同一標(biāo)本采用雙向重復(fù)測(cè)序。3 8-羥基脫氧鳥(niǎo)苷的檢測(cè)及分組:胃癌組織常規(guī)切片4μm,用二甲苯脫蠟后在不同濃度的酒精中水化;在4℃下過(guò)夜孵化,孵化液為1:100的一抗(8-OHd G);滴加生物素化的二抗,室溫下濕盒內(nèi)孵育60min;滴加鏈霉菌抗生物素蛋白-過(guò)氧化物酶溶液,并用新配的DAB液顯色。兩位病理學(xué)專(zhuān)家在400倍光學(xué)顯微鏡下隨機(jī)觀察10個(gè)區(qū)域。根據(jù)8-OHd G陽(yáng)性細(xì)胞所占百分比分為2個(gè)等級(jí):陽(yáng)性細(xì)胞所占百分比≤50%為低表達(dá);陽(yáng)性細(xì)胞所占百分比50%為高表達(dá)。4統(tǒng)計(jì)分析:采用χ2檢驗(yàn)比較計(jì)數(shù)資料,并計(jì)算P值、相對(duì)危險(xiǎn)度(OR)、95%可信區(qū)間(CI)。用Kaplan-Meier法繪制生存曲線,用Log-Rank法分析單因素生存期的差異。實(shí)驗(yàn)數(shù)據(jù)采用SPSS19.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,P0.05被認(rèn)為有統(tǒng)計(jì)學(xué)意義。結(jié)果:1研究已發(fā)現(xiàn)胃癌的發(fā)病風(fēng)險(xiǎn)與線粒體DNA D-Loop區(qū)的單核苷酸多態(tài)性有關(guān)。我們將8-OHd G的不同表達(dá)水平分別與D-Loop區(qū)523-524AC/del、16362T/C、16519C/T及309C/C insert位點(diǎn)進(jìn)行比較分析,結(jié)果顯示在16519位點(diǎn),8-OHd G在C基因型中的表達(dá)水平明顯高于T基因型的表達(dá)(P=0.017),而與523-524AC/del、16362T/C及309C/C insert位點(diǎn)多態(tài)性無(wú)明顯關(guān)系(P0.05)。2檢測(cè)131例胃癌患者術(shù)后胃癌組織中8-OHd G的表達(dá)水平,發(fā)現(xiàn)8-OHd G的免疫反應(yīng)主要發(fā)生在細(xì)胞核,分析其與患者3年生存率之間的關(guān)系,結(jié)果顯示8-OHd G高表達(dá)與低表達(dá)的3年生存率分別為46.81%和46.43%,8-OHd G的表達(dá)水平與患者3年生存率之間無(wú)顯著性差異(P0.05),尚不能說(shuō)明8-OHd G可作為判斷胃癌預(yù)后的獨(dú)立危險(xiǎn)因子。3將8-OHd G表達(dá)水平分別與患者的臨床特征(年齡、性別、臨床分期、腫瘤直徑、腫瘤位置、分化程度、病理類(lèi)型、脈管瘤栓)進(jìn)行分析,結(jié)果顯示8-OHd G的表達(dá)水平與年齡、性別、臨床分期、腫瘤直徑、腫瘤位置、分化程度、脈管瘤栓等均無(wú)明顯關(guān)系(P0.05)。結(jié)論:1前期研究發(fā)現(xiàn)16519C基因型增加了胃癌發(fā)病風(fēng)險(xiǎn),而16519C基因型的DNA氧化損傷標(biāo)志物8-OHd G明顯高于T基因型,16519C基因型可能提高了氧化損傷的水平,從而引起胃癌。2單獨(dú)檢測(cè)8-OHd G的表達(dá)水平尚不能作為判斷胃癌預(yù)后的獨(dú)立危險(xiǎn)因子。3 8-OHd G的表達(dá)水平與年齡、性別、臨床分期、腫瘤直徑、腫瘤位置、分化程度、脈管瘤栓等臨床特征均無(wú)明顯關(guān)系。
[Abstract]:Objective: gastric cancer (gastric, cancer, GC) in the first place in our country's malignant tumor mortality in patients with advanced, easily metastasis, poor prognosis, a serious threat to people's life and health, early diagnosis is the most effective way to improve the prognosis of gastric cancer. In recent years, the relationship between DNA oxidative damage and gastric cancer become a research hotspot. The oxidative damage to proteins, damage the structure and function of biological macromolecules such as lipids and DNA, and thus lead to gene mutation, carcinogenesis and individual aging phenomenon. The mitochondrial DNA (mitochondrial DNA, MT DNA) in a high oxygen environment, damage repair system is not complete, lack of protection group protein, so compared with the nuclear genes are more susceptible to reactive oxygen species (reactive oxygen, species, ROS).D- damage ring area (D-Loop area) is the control center of MT DNA replication and transcription, this area became the MT DNA mutation Hot, our previous study has identified and the risk of gastric cancer related mitochondrial D- loop region linked polymorphic loci, 73G, 16519C and 523-524Del genotype may increase the risk of gastric cancer, 16362C and 309C insert genotype may reduce the risk of gastric cancer, and that these sites may change the level of mitochondrial oxidation the formation of gastric cancer caused by oxygen free radical attack. The product of DNA 8- hydroxydeoxyguanosine (8-hydroxy-2-Deoxyguanosine, 8-OHd, G) during DNA replication can cause G:C-T:A transversion, can be used as exogenous and endogenous factors on the biology of DNA oxidative damage markers. In nasopharyngeal carcinoma, lung cancer, liver cancer, ovarian cancer, malignant tumor cervical cancer, the study found that the expression level of 8-OHd G was significantly higher than that of normal, can be used to evaluate the progress and prognosis of malignant tumors. This study by immunohistochemical method, through The expression level of G 8-OHd in gastric tissue was detected by oxidation damage analysis of oxidation damage associated with the risk of gastric cancer in the mitochondrial DNA D- single nucleotide polymorphism (single nucleotide loop polymorphisms, SNPs) to explore the relationship between the mechanism of the occurrence and development of gastric cancer, and analyze the relationship between oxidative damage and clinical features and prognosis of gastric cancer and provide new ideas for early diagnosis and prognosis of gastric cancer. Methods: 1 subjects, specimen collection and follow-up: from March 2007 -2008 year in August 131 cases of the fourth hospital of Hebei Medical University underwent radical gastrectomy patients preoperatively diagnosed by endoscopy and pathology of gastric cancer were diagnosed. All the patients were not for the clinical treatment of chemotherapy and / or radiotherapy and postoperative pathological results were confirmed by two or more experience of Pathology, the fourth hospital of Hebei Medical University. The doctor diagnosed rich details Detailed records of all patients with gender, age, tumor location, tumor size and clinical stage of clinical features, through outpatient review, letters or phone on postoperative patients with gastric cancer survival were followed up. The follow up deadline is August 2011. Postoperative gastric cancer tissues were collected after the fourth hospital of Hebei Medical University ethics committee approved.2 mitochondrial DNA amplification and DNA sequencing. To extract the identification of qualified DNA after amplification by PCR, primer: 5'-CCCCATGCTTACAAGCAA-GT-3', primer: 5'-GCTTTGAGGAGGTAAGCTAC-3'. amplified by agarose gel purified and sequenced, the instruments for the ABI PRISM#174; 3730XL sequencing. The same samples were used for detection and sequencing of.3 8- two-way repeated packet hydroxydeoxyguanosine: Gastric cancer tissue paraffin sections of 4 m, after dewaxing with xylene in different concentrations of alcohol and water at 4 DEG C; Under overnight incubation, hatching solution for 1:100 - (8-OHd G); dropping biotinylated anti two, room temperature wet box incubated in 60min; adding streptavidin peroxidase solution, and DAB solution with a new color. Two pathologists in 400 times under the light microscope were observed in 10 regions. According to the percentage of 8-OHd G positive cells were divided into 2 grades: the percentage of the positive cells less than 50% for low expression; the percentage of the positive cells of 50% high expression of.4: statistical analysis using chi square test to compare the 2 count data, and calculate the P value, relative risk (OR), 95% confidence interval (CI). The survival curve was drawn by Kaplan-Meier method, difference analysis of single factor survival by Log-Rank method. The experimental data were analyzed by SPSS19.0 statistical software. P0.05 was considered statistically significant. Results: 1 studies have found that the risk of gastric cancer and mitochondrial D Single nucleotide polymorphisms in NA D-Loop area. We will 8-OHd G D-Loop respectively with different expression levels of 16362T/C, 16519C/T and 523-524AC/del, 309C/C and insert loci were analyzed, the results show that among the 16519 loci, the expression level of 8-OHd G expression in C genotype was significantly higher than that of the T genotype (P=0.017), and 523-524AC/del no obvious relationship between 16362T/C and 309C/C, the polymorphism of insert (P0.05).2 expression level was detected in 131 cases of patients with gastric cancer in gastric cancer 8-OHd G, found that the immune response of 8-OHd G occurred mainly in the nucleus, analyze the relationship between the 3 year survival rate of the patients and showed high 8-OHd expression and low expression of G the 3 year survival rates were 46.81% and 46.43%, there was no significant difference between the 3 year survival rate of patients with 8-OHd and the expression level of G (P0.05), still can not demonstrate that 8-OHd G can be used as an independent risk of gastric cancer after pre judgment The 8-OHd factor.3 G expression level and clinical characteristics of patients (age, gender, clinical stage, tumor size, tumor location, degree of differentiation, pathological type, tumor thrombus) were analyzed, the results showed that the expression level of 8-OHd G and age, gender, clinical stage, tumor size, tumor location, degree of differentiation. There was no significant correlation between vascular tumor emboli (P0.05). Conclusion: 1 the previous study found that 16519C genotype increased the risk of gastric cancer, and 16519C genotype DNA oxidation injury markers 8-OHd G was significantly higher than T genotype, 16519C genotype may increase the level of oxidative damage, causing gastric cancer to detect the expression of.2 alone 8-OHd G still can't be judged as expression and age, the prognosis of gastric cancer.3 8-OHd G independent risk factors of gender, clinical stage, tumor size, tumor location, degree of differentiation, clinical features of vascular invasion were not significantly Relationship.

【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】：2015
【分類(lèi)號(hào)】：R735.2

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