【作者】 向燕；

【导师】 王远亮；

【作者基本信息】 重庆大学 ， 药物化学， 2005， 硕士

【摘要】 肝癌是我国常见的恶性肿瘤之一,是我国位居第二的癌症“杀手”,但现在还缺乏治疗肝癌的有效药物,研究筛选抗肝癌的药物是我国面临的重要抗癌任务。在分子肿瘤学取得迅速进展的今天,抗癌药物的筛选将由针对疾病的筛选转向针对机制的筛选,其关键是寻找可供治疗干预的特殊分子靶点,也就是找到正常细胞与癌细胞之间的生化与分子差异。因此,如果对肿瘤发生和发展的分子机制进行深入研究,试图找到一种全面分析评价抗肿瘤药物对肿瘤作用机制的方法,将为抗肿瘤药物的筛选提供一个新的策略。 本文利用LongSAGE技术,定性和定量研究丹皮酚胂化物2-甲氧基-4-羟基-5-乙酰基偶氮苯-4-胂酸（R₂）对人肝癌细胞HepG2基因表达差异的影响,寻找其作用的分子靶点,探讨丹皮酚胂化物抗肝癌作用的分子机制。 以丹皮酚胂化物R₂作用的人肝癌HepG2细胞作为研究对象,提取总RNA,用生物素的磁珠逆转录合成双链的cDNA,用锚定酶Nla Ⅲ酶切cDNA,将酶切的cDNA片段分成两份,分别与连接子A、B连接。用标签酶Mme Ⅰ酶切连接子和cDNA结合的序列,释放出带有连接子的标签,使用T4 DNA连接酶连接带有连接子A、B的标签,形成130bp的双标签,并进行大规模PCR扩增。锚定酶Nla Ⅲ切除连接子A、B,得到34bp双标签,再把双标签连接成串联体,最后将串联体克隆转化测序。用SAGE2000软件对序列进行分析,获得丹皮酚胂化物作用人HepG2细胞的LongSAGE标签库。将构建好的LongSAGE标签库与SAGEmap中人的肝癌组织的SAGE标签库进行比对,从而了解R₂作用后人肝癌细胞基因表达差异的情况。 从构建好的LongSAGE标签库中挑选克隆45个,进行测序,测序一共得到标签675个,特异性标签为401个,其中单一匹配标签263个,占66%,多重匹配标签57个,占14%,新标签81个,占20%。在构建好的LongSAGE标签库与人的肝癌的SAGE标签库之间,基因表达存在明显差异,主要差异表达的基因有PAFAH2、MCM3、CTPS、GFM2、HSP70、TMSB4X、TXNRD1、CYP3A4、MAP2K2、SET、CASP8、OXCT1等。结果表明,R₂通过下调了PAFAH2、MCM3、CTPS、GFM2、HSP70的表达,上调了TMSB4X、TXNRD1、CYP3A4、MAP2K2、SET、CASP8、OXCT1的表达而发挥着抗肝癌的作用。 SAGE方法能较完整地、定量地获得基因表达信息,识别丹皮酚胂化物抗肝癌作用的分子靶点,有助于阐明丹皮酚胂化物作用的分子机制,进一步表明丹皮酚胂化物有可能成为一种新的抗肿瘤药物。更多还原

【Abstract】 Hepatocellular carcinoma(HCC) is one of the most common malignant tumors, and has become the second leading killer in cancer. There is a lack of efficient drugs for HCC therapy, so discovery and screening new drugs for the treatment of liver cancer is essential and important. In now days with the rapid development of molecular oncology, the screening of anticancer drugs is converted from that aiming at diseases to that aiming at mechanism. It is key in search of specific molecular targets for therapy, i.e. discovery of the biochemical and molecular difference between cancer and normal cells. Therefore, if in deep research of the mechanism of hepatocarcinogenesis, it will try to find out the method of analysis and estimate of action mechanism of anti-cancer drugs, providing a new strategy for the screening of anti-cancer drugs.In this study, the genes differentially expressed in human hepatoma cell line HepG2 induced by paeonol-arsenic compound, 2-methoxy-4-hydroxy-5-acetyl-azobenzene -4-arsenic acid (R2), were analyzed qualitatively and quantitatively with long serial analysis of gene expression (LongSAGE) protocol, and then the genes expression-profile was.established. We try to find out the molecular targets, and discuss the molecular mechanism of paeonol-arsenic compound acting on hepatocellular carcinoma.Total RNA was isolated from HepG2 induced by paeonol-arsenic compound. The cDNA was synthesized from mRNA immobilized on magnetic beads. The cDNA was then cleaved with a restriction enzyme called NlaIII, and the cleavage product was divided equally into two portions. Adapter A and Adapter B were ligated to each portion using Nlalll cohesive termini, then the tags were released with digestion of tagging enzyme Mme I. The tags linked together to establish the 130bp ditags by T4 DNA ligase. The products were scale-up PCR-amplified to obtain adequate 130bp ditags, and were cleaved with Nlalll again to produce 34bp ditags. The 34bp ditags were ligated to obtain concatemers. The concatemers were cloned into pZErO(?)-1 vector, transformed and ready to sequence. The raw sequencing files were analyzed by SAGE2000 software, and a LongSAGE library was constructed from HepG2 induced by paeonol-arsenic compound. The LongSAGE library was compared with hepatocellular carcinoma (HCC) library from the SAGEmap, in order to find out the differentially expressed genes.A total of 675 tags were extracted from the 45 the sequenced files, representing401 unique tags. Of these tags, 263 (66%) tags occurred once and 57 (14%) tags were identified multiple times. 81 (20%) tags were not reported before. Between the LongSAGE library and HCC library, there were much difference of gene expression, such as PAFAH2、 MCM3、 CTPS、 GFM2、 HSP70、 TMSB4X 、 TXNRD1、 CYP3A4、 MAP2K2、 SET、 CASP8、 OXCT1 et al. The results indicate that R2 can down-regulate the expression of PAFAH2、MCM3、CTPS、GFM2、HSP70, up-regulate the expression of TMSB4X、 TXNRD1、 CYP3A4、 MAP2K2、 SET、 CASP8、 OXCT1. It is also suggested that R2 may play important roles in the anti-tumor effect.SAGE can provide the full and quantitative information of gene expression, identify the molecular targets of paeonol-arsenic compound, and help to clarify the molecular mechanism of paeonol-arsenic compound. It further indicates that paeonol-arsenic compound may be a potential anti-tumor drug.更多还原