胃癌相关性线粒体D-环多态位点与8-羟基脱氧鸟苷表达的关系

发布时间：2018-01-03 11:22

本文关键词：胃癌相关性线粒体D-环多态位点与8-羟基脱氧鸟苷表达的关系　出处：《河北医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:胃癌(gastric cancer,GC)在我国恶性肿瘤的死亡率中居首位,就诊患者以中晚期为主,易转移复发,预后差,严重威胁了人们的生命健康,早期诊断是改善胃癌预后的有效方法之一。近年来,DNA氧化性损伤与胃癌发病的关系成为了研究热点。氧化损伤会对蛋白质、脂类及DNA等生物大分子的结构和功能造成损伤,并由此导致基因突变、细胞癌变及个体衰老等现象。线粒体DNA(mitochondrial DNA,mt DNA)处于高氧环境中,损伤修复系统不完整,缺乏组蛋白的保护,因此与核基因相比更易受到活性氧(reactive oxygen species,ROS)的损伤。D-环区(D-Loop区)是mt DNA分子复制及转录的调控中心,此区域成为了mt DNA突变热点,我们前期研究已经鉴定了和胃癌发病风险相关联的线粒体D-环区多态位点,73G、16519C及523-524Del基因型可能增加了胃癌的发病风险,16362C及309C insert基因型则可能降低了胃癌的发病风险,并推论这些位点可能改变了线粒体的氧化水平而引起胃癌。氧自由基攻击DNA所形成的产物8-羟基脱氧鸟苷(8-hydroxy-2-Deoxyguanosine,8-OHd G),在DNA复制时可引起G:C-T:A颠换突变,可作为外源性及内源性因素对DNA氧化损伤的生物学标志物。目前在鼻咽癌、肺癌、肝癌、卵巢癌、宫颈癌等恶性肿瘤中,研究发现8-OHd G的表达水平明显高于正常,可用于评估恶性肿瘤的进展及预后。本研究通过免疫组织化学的方法,通过检测胃癌组织中8-OHd G的表达水平以反应氧化损伤的程度,分析氧化损伤与胃癌发病风险相关的线粒体DNA D-环区单核苷酸多态性(single nucleotide polymorphisms,SNPs)之间的关系,以探索胃癌的发生发展机制,并分析氧化损伤与胃癌预后及临床特征的关系,为胃癌的早期诊断及预后监测提供新思路。方法:1研究对象、标本采集及随访:选取2007年3月-2008年8月于河北医科大学第四医院外三科行胃癌根治术的131例患者,术前均经内镜检查及组织学病理确诊为胃癌。所有患者术前均未行化疗和/或放疗等临床治疗,术后病理结果均经河北医科大学第四医院病理科两名以上经验丰富的医师确诊。详细记录所有患者的性别、年龄、肿瘤部位、肿瘤直径及临床分期等临床特征,通过门诊复查、信件或电话对胃癌患者术后生存状况进行随访,随访截止时间为2011年8月。术后胃癌组织的收集均经过河北医科大学第四医院伦理委员会批准。2线粒体DNA扩增及测序:将已提取的鉴定合格后的DNA,采用PCR技术扩增目的片段,上游引物为:5'-CCCCATGCTTACAAGCAA-GT-3',下游引物为:5'-GCTTTGAGGAGGTAAGCTAC-3'。扩增产物经琼脂糖凝胶纯化后,进行测序,所用仪器为ABI PRISM#174;3730XL测序仪。同一标本采用双向重复测序。3 8-羟基脱氧鸟苷的检测及分组:胃癌组织常规切片4μm,用二甲苯脱蜡后在不同浓度的酒精中水化;在4℃下过夜孵化,孵化液为1:100的一抗(8-OHd G);滴加生物素化的二抗,室温下湿盒内孵育60min;滴加链霉菌抗生物素蛋白-过氧化物酶溶液,并用新配的DAB液显色。两位病理学专家在400倍光学显微镜下随机观察10个区域。根据8-OHd G阳性细胞所占百分比分为2个等级:阳性细胞所占百分比≤50%为低表达;阳性细胞所占百分比50%为高表达。4统计分析:采用χ2检验比较计数资料,并计算P值、相对危险度(OR)、95%可信区间(CI)。用Kaplan-Meier法绘制生存曲线,用Log-Rank法分析单因素生存期的差异。实验数据采用SPSS19.0统计软件进行统计学分析,P0.05被认为有统计学意义。结果:1研究已发现胃癌的发病风险与线粒体DNA D-Loop区的单核苷酸多态性有关。我们将8-OHd G的不同表达水平分别与D-Loop区523-524AC/del、16362T/C、16519C/T及309C/C insert位点进行比较分析,结果显示在16519位点,8-OHd G在C基因型中的表达水平明显高于T基因型的表达(P=0.017),而与523-524AC/del、16362T/C及309C/C insert位点多态性无明显关系(P0.05)。2检测131例胃癌患者术后胃癌组织中8-OHd G的表达水平,发现8-OHd G的免疫反应主要发生在细胞核,分析其与患者3年生存率之间的关系,结果显示8-OHd G高表达与低表达的3年生存率分别为46.81%和46.43%,8-OHd G的表达水平与患者3年生存率之间无显著性差异(P0.05),尚不能说明8-OHd G可作为判断胃癌预后的独立危险因子。3将8-OHd G表达水平分别与患者的临床特征(年龄、性别、临床分期、肿瘤直径、肿瘤位置、分化程度、病理类型、脉管瘤栓)进行分析,结果显示8-OHd G的表达水平与年龄、性别、临床分期、肿瘤直径、肿瘤位置、分化程度、脉管瘤栓等均无明显关系(P0.05)。结论:1前期研究发现16519C基因型增加了胃癌发病风险,而16519C基因型的DNA氧化损伤标志物8-OHd G明显高于T基因型,16519C基因型可能提高了氧化损伤的水平,从而引起胃癌。2单独检测8-OHd G的表达水平尚不能作为判断胃癌预后的独立危险因子。3 8-OHd G的表达水平与年龄、性别、临床分期、肿瘤直径、肿瘤位置、分化程度、脉管瘤栓等临床特征均无明显关系。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

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