【作者】 潘敏；

【导师】 祁鸣；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2012， 博士

【摘要】 第一部分：中国人乳腺癌相关基因BRCA1/2基因的突变检测及LOVD数据库构建目的：作为国际基因变异组计划的一部分(HVP-China),利用LOVD格式,构建一个针对中国人群的BRCA1/2基因变异新数据库。方法：搜索PubMed以及一些中文数据库如万方、维普等,收集所有在中国人群中报道过的BRCA1/2基因的变异,将其以正确的格式提交到LOVD数据库。此外,在临床基因检测过程中,收集家族性乳腺癌和早发性乳腺癌患者的外周血样本,提取DNA,利用PCR和测序检测BRCAl基因的变异,并将新发现的变异提交到数据库。结果：到目前为止,根据我们数据库中的内容,在中国人的BRCA1和BRCA2基因上已经分别发现了136和62个不同的变异。与其他种族的人群相比,中国人有其独特的BRCA1/2基因突变热点和高频突变。在这两个基因上的14个最常见的致病突变中,有50%是BIC数据库中未报到过的。结论：在中国人群中建立了第一个BRCA1/2基因变异数据库。BRCA1/2基因在中国人中的突变与其他种族的人群有所不同。该数据库的建立有助于科研工作者和临床医生对遗传性乳腺癌和卵巢癌以及其它由BRCA1/2基因突变引起的肿瘤的研究、检测和诊断,尤其有利于今后中国人群的BRCA1/2基因检测。第二部分：DTWD1基因在胃癌中的功能和调控研究目的：明确DTWD1基因在胃癌细胞中的功能,寻找其可能的调控因子以及其调控机制。方法：在正常胃细胞和胃癌细胞系中、TSA处理前后的胃癌细胞中以及临床手术收集的正常和胃癌组织中,用实时定量RT-PCR检测DTWD1的表达水平。用不同的HDAC siRNA转染胃癌细胞,以明确DTWD1的表达主要是受到哪一个HDAC的抑制。荧光素酶报告基因分析用于确定DTWD1基因的启动子区域,然后用ChIP检测p53蛋白和DTWD1启动子的结合情况。为了验证DTWD1的基因的抑癌功能,细胞在转染DTWD1表达载体或siRNA后进行MTS和克隆形成试验。Western blot用于检测蛋白表达水平。结果：DTWD1在胃癌中表达下调,TSA处理或转染HDAC siRNA能上调其表达。DTWD1在胃癌中的下调主要是受到HDAC3的抑制。P53蛋白能够与DTWD1启动子结合并促进其表达,而HDAC3会影响两者的结合作用。在胃癌细胞中过表达外源DTWD1能够抑制细胞的生长和克隆形成能力,而在正常细胞中用siRNA敲除该基因后,细胞生长速度则加快。结论：DTWD1在胃癌中起着抑癌基因的功能,它的表达受到HDAC3的抑制,而p53能促进其表达。更多还原

【Abstract】 Part one:Mutation detection and LOVD Database for BRCA1/2Gene in the Chinese PopulationObjective:To created a novel variants database for BRCAI and BRCA2in the Chinese population using the Leiden Open Variation Database (LOVD) format, as part of the International Variome Project (HVP-China)Methods:We searched PubMed and some Chinese searching engines to collect all the variants of BRCAI and BRCA2in the Chinese population that have been reported. In addition, peripheral blood samples of the patients with familiar or early-onset breast cancer were colleted. DNA was abstracted, PCR and sequencing were used for detection of BRCAI gene. The novel variation observed in the gene test was also submitted to our LOVD database.Results:To date, our database includes136unique variants of BRCAI and62unique variants of BRCA2. There are some differences in BRCA1/2gene mutations between Chinese population and that of other ethnicities. Of the14pathogenic mutations reported most frequently,50%cannot be found in the Breast Cancer Information Core database.Conclusion:we have created the first known variation database of BRCA1/2in Chinese population. There are some differences in BRCA1/2gene mutations between Chinese population and that of other ethnicities. This database will provide a great convenience to researchers and clinicians who study, test, and diagnose Hereditary breast cancer and ovarian cancer (HBOC) and other cancers caused by mutations on BRCAI and BRCA2. This database can be of especial use for facilitating future genetic tests of these two genes in Chinese population.Part two:Function and Regulation of DTWD1Gene in Gastric CancerObjective:To establish the function of DTWD1, as well as the potential regulation factor and mechanism of this gene in gastric cancer cells.Methods:DTWD1expression level was detected by quantitative real-time RT-PCR in gastric cancer cell lines with or without TSA treatment, normal gastric cell line (GES), and normal or tumor samples from clinical surgery. The cells were transfected with different HDAC siRNAs to determine which HDAC contributes most to DTWD1dysregulation. Luciferase assay was used to determine the promoter region of DTWD1gene and ChIP was used to examine the binding of p53protein and DTWD1promoter. To assess the tumor-suppressor function of DTWD1, MTS assay and colony formation assay were carried out in cells transfected with DTWD1expression vector or siRNA. Western blot was used to detect protein expression level.Results: DTWD1is downregulated in gastric cancer and can be upregulated by TSA treatment. HDAC3is the most important HDAC which contributes to DTWD1down regulation in gastric cancer cell lines.The protein p53can bind to the promoter of DTWD1and p53is a positive regulator of DTWD1. HDAC3impacts the binding of p53protein and DTWDlgene. Ectopic DTWDl expression inhibits tumor cell growth and colony formation ability.Conclusion:DTWDl is a novel gene which functions as a tumor suppressor in gastric cancer. DTWDl was regulated through HDAC3repression and p53activation.更多还原