【作者】 王建；

【导师】 沈建根；

【作者基本信息】 浙江大学 ， 免疫学， 2003， 硕士

【摘要】 氧化剂作为线粒体电子转移、炎症反应、有氧代谢过程的副产品，在体内不断产生，尽管机体的酶促和非酶促抗氧化防御体系可保护细胞免受氧化损伤，但仍有一些氧化剂逃避了机体的防御机制而造成大分子损伤，包括蛋白质、脂质、DNA、RNA。DNA的氧化损伤与老化，年龄相关的退行性疾病以及肿瘤的发生有着密切关系。 oh~8dG是活性氧导致的一个DNA碱基修饰产物，在DNA复制过程中，oh~8dG可引起G：C→T：A的颠换，从而引起突变。1986年Floyd等利用高效液相色谱电化学法测定了oh~8dG，这使得测定细胞DNA中的oh~8dG成为可能。oh~8dG作为DNA氧化损伤重要的生物标记物，受到广泛重视。 本研究以BSA-oh~8dG偶联物免疫BALB/C小鼠，用杂交瘤技术制备抗oh~8dG的单克隆抗体，通过酶标记技术检测oh~8dG在组织和细胞中的表达，建立一种快速、敏感、特异而简便的oh~8dG检测方法，探讨oh~8dG在疾病的发生和发展过程中所起的作用。 1．BSA-oh~8dG偶联物的制备：oh~8dG单体和BSA按1：4的比例混匀，加入碳二亚胺，避光，4℃搅拌过夜，用生理盐水透析平衡，浓缩至1ml体积，加50％甘油-20℃保存，即制成BSA-oh~8dG偶联物，BSA-G按同样方法制备。 2．抗oh~8dG单克隆抗体的制备：首次免疫，将BSA-oh~8dG以弗氏完全佐剂乳化，第2、3次免疫以弗氏不完全佐剂乳化，背部皮内多点注射，每周1次，每次0.2ml(含偶联物60μg)，1月后加强免疫1次，4天后取脾细胞与SP2/0细胞在50％聚乙二醇作用下融合，经HAT选择性培养。2周后用ELISA间接法筛 浙江大学硕士研兜生论文选阳性杂交瘤，选择抗BSA七h勺G阳性而抗BSA－G、BSA阴性的杂交瘤细胞，并予以有限稀释克隆化培养，直到克隆化细胞抗体阳性率为 100％。杂交瘤细胞接种经液体石蜡预处理过的BALB／C小鼠腹腔，两周后收集腹水，离心后取上清，－20C保存。ELISA测定腹水效价为 1：16384，Western blot证实该单抗有较好的特异性，竞争性ELISA测定单抗的交叉反应率＜10％。 3 胃粘膜组织。h勺G测定：以单克隆抗体作免疫组化检测。h勺G在慢性浅表性胃炎组织中的表达，免疫组化结果显示，20例Hp阳性的慢性浅表性胃炎组织中有 11例为阳性表达，阳性率为 55％；20例 Hp阴性的慢性饯表性胃炎组织中洲、G的阳性表达率为 5O，Hp阳性和 Hp阴性胃粘膜组织之问的讪勺G表达水平有显著性差异。 4 HL七0细胞中ohsdG的检测：将生长良好的HL七0细胞调整至50Xm’偷l，分成4组，其中3组加入H。O。，使之终浓度为4mmow，另外一组作对照，分别于4h、6h、gb取细胞涂片（载玻片预先用多聚赖氨酸处理），镜下检查细胞密度 以排列较密但不重叠为宜，待涂片略于后用冷丙酮固定15min，取出自然干燥后，进行免疫细胞化学检测。结果显示，在正常的 HL七0细胞中，加勺G表达最低，HL七0细胞经过氧化氢处理6h，Oh勺G表达最高，不同阶段的加勺G表达水平有 显著性差异（Radii分析，P＜005）。 5 抗 Oh勺G单克隆抗体酶联免疫检测法的建立：以不同浓度的加勺G标准 品作为检测标本，绘制标准曲线，建立了检测。h‘dG的竞争性ELISA。结果表 明，该方法特异、敏感门0n旮ml入重复性好傈异系数＜10％人 结论： 1．制备的抗汕勺G的单克隆抗体能特异识别讪勺G。 2 Hp感染可使胃粘膜中的。h勺G水平升高，并且阳性表达主要在胞浆， 。h勺G作为一突变剂，可能在 Hp感染引起的胃癌中起着重要作用。 3、HL七0经外源性过氧化氢作用后，。h勺G表达水平增高，不同阶段的 。h‘dG表达水平有显著性差异。 4 竟争性ELISA具有灵敏度高、特异性强的特点，可望成为临床检测。hsdG 的试剂盒。更多还原

【Abstract】 Oxidant are generated continuously as normal byproduct of mitochondrial electron transport ,inflammatory reactions,and many other processes associated with aerobic metabolism. Although elaborate enzymatic and nonenzymatic defences exist that protect the cell against oxidant-induced damage ,the oxidant that escape the defences damage celluar macromolecules including protein, lipid, DNA and RN A . Unrepaired oxidative damage to DNA,for example ,has been proposed to be a major contributor to aging and other age-related degenerative diseases including cancer.8-hydroxy-2’-deoxyguanosine, a DNA-base modified product generated by reactive oxygen species ,has the biologic significance of inducing G:C→T:A transversions at DNA replication. In 1986,Floyd reported a sensitive detection method for oh8dG detection by an electrochemical detector connected with HPLC This made the detection of oh8dG possible in cellular DNA.In this study, the monoclonal antibody against oh8dG was prepared by hybridoma technique The specificity of McAb was confirmed by competitive ELISA and Western blot. We succeeded in establishing an immunohistochemistry method to detect oh8dG in gastric mucosa and HL-60 cell , and oh8dG was detected in HL-60 cell by competitive ELISA. This will help us to know the function of oh8dG in the development of disease.1. The preparation of BSA-oh8dG conjugate : oh8dG and BSA mixed together with the proportion of 1:4, the mixture was milled at 4℃ overnight . Then it was dialysed with normal saline and stored at -20癈 with 50% glyceral.2. The preparation of monoclonal antibody against oh8dG.For the firstimmunization BSA-oh8dG was emulsified by CFA , it was emulsified by IFA in the second and third immunization .BALB/C mice were injected subcutaneously at two dorsal sites with 200ul conjugate. One month later, mice were immunized with BSA-oh8dG. Four days later ,hybridoma cell were prepared . Then the supernatant was detected to select the positive cell which was cloned by limiting dilution method, until the positive rate is up to 100%.Western blot showed this McAb was fairly specific for oh8dG and competitive ELISA indicated its cross reactivity was less than 10%.3. The detection of oh8dG in gastric mucosa : The positive rate in gastric tissues with Hp infection and without Hp infection was 55%, 5% respectively(P<0.01) . Oh8dG was mainly present in cytoplasm and few in nucleolus.4. The detection of oh8dG in HL-60 cell: The cell is adjusted to 5.0 × 105/ml ,then it was divided into four flasks and each contains five millilitres . H2O2 was added to three flasks and the final concentration is 4mmol/L, the other flask was used as a control . The cells were painted on a slide which had been treated with polylysine. When the slide dried ,it was fixed with acetone for 15 min and used for immunocytochemistry. The results indicated that H2O2 increased oh8dG expression in HL-60 cell and oh8dG was obviously differential expression in different stages.5. Establishment of competitive ELISA method: Standard samples of different concentration were used to detect oh8dG . The results showed that this method was highly sensitive(10ng/ml) with the reproducibility(CV<10%) . Conclusion:1. This monoclonal antibody can specially recognize oh8dG2. Hp infection can increase oh8dG content in gastric mucosa .As a mutagen, oh8dG probably play an important role in the carcinogenesis of stomach.3. Hydrogen peroxide increased oh8dG expression in HL-60 cell and oh8dG was obviously differential expression in different stages.4. Competitive ELISA showed that the advantage of the method lied in its sensitivity(10ng /ml),reproducibility(CV< 10%).更多还原