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1、上海交通大学 博士学位论文 DNA甲基化酶蛋白在人胃癌组织中的表达及其临床意义 姓名：丁雯瑾 申请学位级别：博士 专业：内科学 指导教师：房静远;陈晓宇 20070425 上海交通大学医学院八年制博士论文 I DNA 甲基化酶蛋白在人胃癌组织中 的表达及其临床意义 甲基化酶蛋白在人胃癌组织中 的表达及其临床意义 摘 要 摘 要 目的 目的 消化道肿瘤发病率高, 危害性大。大量研究业已证明包括胃癌 在内的恶性消化道肿瘤，其发生发展是多因素、多步骤的过程，其中DNA 甲基化在基因表达调控中具有重要的作用。DNA甲基化是一个可逆性过 程：一方面DNA甲基化酶(DNA methyltranferase
2、,Dnmt)催化甲基基团转 移至胞嘧啶参与甲基化的形成(主要是Dnmt3a和Dnmt3b)和维持（主要是 Dnmt1） ；另一方面去甲基化酶又起着与Dnmt相反的作用。我们拟使用免 疫组化方法分析胃癌组织标本中Dnmts的表达情况， 并探讨其与患者临床 情况的关系。 明确1） 胃癌、 癌旁及其正常组织中的DNA甲基化酶 （Dnmt1、 Dnmt3a 和Dnmt3b）表达有何差异。2)胃癌组织中Dnmt蛋白表达差异是否 与其生物学行为有关及其临床意义。 方法 方法 收集我院外科手术并经病理证实为胃癌的患者 38 例， 每例标 本分别取癌、癌旁和外周正常黏膜组织。所有标本经福尔马林固定，石 蜡包埋
3、，连续切片 4 张。其中 1 张切片用于 HE 染色，由病理医师进行 上海交通大学医学院八年制博士论文 II 胃癌组织学分类。其余 3 张通过链霉菌抗生物素蛋白-过氧化酶试剂盒 (SP 法)检测组织中 Dnmt1，Dnmt3a 和 Dnmt3b 蛋白表达。观察该三种抗 体在胃黏膜组织中的表达情况，及不同类型的胃癌组织中表达量的差 异。最后通过 SPSS 分析软件进行统计处理，并分析以上免疫组化结果 与胃癌患者一般资料、淋巴结转移的关系。该课题的设计和实施得到了 上海交通大学医学院附属仁济医院医学伦理委员会的批准。 结果 结果 我们的研究表明：1）胃癌组织中 Dnmt1、Dnmt3a、Dnmt3
4、b 蛋白表达的阳性率分别为 81.6%、 81.6%和 68.4%， 显著高于相应的癌旁 组织（39.5%、50和 44.7%）与正常组织（10.5%、10.5%和 7.9%） ， P0.05。 2） 阳性切片可见 Dnmt1 弥散分布于肿瘤或腺体细胞浆与胞核， 染色较均匀。而 Dnmt3a、Dnmt3b 主要分布于肿瘤或腺体细胞浆，色棕 黄，染色均匀。3）Dnmt1 与 Dnmt3a 蛋白表达显著相关(P0.05)；Dnmt3a 与 Dnmt3b 间有 一定相关性呈低度相关(P0.05)。4）Dnmt1 蛋白阳性程度与年龄、淋巴 结转移不相关，Dnmt3 家族与患者年龄、性别、肿瘤分化程度和
5、淋巴结 转移均无明显相关。Dnmt1 与性别、组织生物学行为可能存在关联。 结论结论 我们的研究结果表明：1）胃癌存在 Dnmts 过表达，提示其在 胃癌的发生和发展中起有重要作用。Dnmt1 蛋白主要存在于胞浆和胞 核，Dnmt3 家族存在于胞浆。Dnmt1、3a 蛋白表达量高于 Dnmt3b，提 示在胃肿瘤发生过程中， 前两者可能作用更甚。 与 Dnmt3b 相比， Dnmt3a 上海交通大学医学院八年制博士论文 III 在从头甲基化中可能起着更重要作用。2）Dnmt3 家族与患者年龄、性 别、肿瘤分化程度和淋巴结转移均无明显相关。Dnmt1 与性别、组织生 物学行为可能存在关联， 提示检
6、测其表达量可能对判断患者预后具有一 定意义。 关键词 关键词 胃癌，DNA 甲基化酶，免疫组化，组织生物学行为 上海交通大学医学院八年制博士论文 IV THE EXPRESSION AND CLINICAL SIGNIFICANCE OF DNA METHYLTRASFERASES PROTEIN IN HUMAN GASTRIC CANCER ABSTRACT Objective Gastrointestinal (GI) tumors have high incidence and harms to human beings. A large number of studies have
7、shown tumorigenesis involves many factors and steps. Aberrant DNA methylation is one of the most consistent epigenetic changes in human tumors. It is catalyzed by the three known active DNA methyltransferases(Dnmts) as follows: Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 maintains DNA methylation while the othe
8、r two participant in DNA methylation formation (de novo methylation). Immunohistochemistry offers a good way to locate genetic products like protein in tissues or cells. We adopted it to detect and discuss these two issues: 1)Whether the different expression of Dnmts proteins was related to gastric
9、biological behaviors; 2)Whether three kinds of Dnmts expressed differently in cancerous, para-cancerous and non-cancerous tissues. Methods 114 paired mucosal tissue samples of primary gastric cancer (GC) and corresponding para-cancerous, non-cancerous gastric mucosa tissues were obtained from surgic
10、ally resected specimens of 38 patients. Four five-micrometer-thick sections of each specimen were fixed by formalin and embedded by paraffin. One section was used for HE staining and classified by senior pathologists. The other three were used to detect Dnmts expression by immunohistochemical staini
11、ng. The expression of Dnmt1, Dnmt3a and Dnmt3b in cancerous tissues as well as their relationship with biological behavior were observed. Standard statistical test was carried out by SPSS. 上海交通大学医学院八年制博士论文 V Results We found 1) the positive rates of Dnmt1, Dnmt3a and Dnmt3b in GC tissues were 81.6%,
12、 81.6% and 68.4%, respectively. They were significantly higher than those of para-cancerous tissues (39.5%, 50% and 44.7%) as well as those of normal tissues (10.5%, 10.5% and 7.9%), P0.05. 2) Dnmt1 was well-distributed in cytoplasm and nuclear of tumor cells or glands while Dnmt3a and 3b only in cy
13、toplasm, showing dark brown. 3) There was a significant correlation between Dnmt1 and Dnmt3a proteins (P0.01), a low correlation between Dnmt3a and Dnmt3b (P0.05). 4) Dnmt1 protein expression had no correlation with age and lymphnode metastasis and had possible correlation with gender and biological
14、 behaviors. Dnmt3 family were not related to age, gender, tumor differentiation and lymphnode metastasis. Conclusion Our results indicated: 1) Dnmts overexpressed in GC, involving in gastric tumorigenesis. Dnmt1 was located in cytoplasm and nuclear while Dnmt3 family in cytoplasm only. The expressio
15、n of Dnmt1 and Dnmt3a was higher than that of Dnmt3b. It meant the former two might play a more important role in tumorigenesis. Compared with Dnmt3b, Dnmt3a had more effects on de novo methylation. 2) There was no correlation between Dnmt3 family and age, gender, tumor differentiation and lymphnode metastasis and possible relation between Dnmt1 and gender as well as biological behavior, which meant that the expression of Dnmts protein might play an important role in prognosis. KEY WORDS human gastric cancer, DNA methyltransferase (Dnmt), immunohistochemistry, biological b
