【作者】 彭亮；

【导师】 蒋继志；

【作者基本信息】 河北大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 前列腺癌为全世界特别是欧美国家男性常见的恶性肿瘤和主要死亡原因之一。我国随着人口老年化及医疗检测水平的提高，其发病率逐年增加。按照近年来全国前列腺癌的发病率统计，前列腺癌被列为我国21世纪可能增长最快的肿瘤之一。 我们在前期实验中利用基因表达系列分析(Serial analysis of gene expression，SAGE)数据库挖掘的方法发现基因PCa05在前列腺癌组织的表达数据库中高强度表达，超过癌周围正常组织的10倍。同时在人体所有肿瘤及正常组织中，PCa05在前列腺癌组织的表达强度最高。用Northern Blot初步证实了PCa05为前列腺癌上调表达的相关癌基因。通过基因功能检索，我们没有发现任何关于PCa05功能研究的报道。 根据PCa05序列合成一对两端带有限制性酶切位点的引物，提取细胞的总RNA后进行RT-PCR扩增，以Hind Ⅲ和EcoR Ⅰ双酶切分别切取真核表达载体pEGFP—C1和目的基因PCa05片段，然后将两者连接起来，从而获得重组质粒。经酶切及测序鉴定后，以脂质体转染试剂Lipofectamin 2000将构建好的重组质粒转染至培养的LNCaP细胞中，Northern Blot初步证明了融合基因的表达，然后进行细胞爬片，24h后放置在荧光共聚焦显微镜下观察融合蛋白的亚细胞定位，同时以生物信息学方法对基因PCa05进行序列分析及功能预测。 初步得到了基因PCa05的功能预测信息，基因PCa05可能是一个受oct-1等转录因子调控、主要在细胞线粒体发挥基因调节作用的可溶性蛋白；成功的构建了pEGFP-C1-PCa05融合蛋白的真核表达载体，融合蛋白在细胞核及细胞浆均有表达。与单纯EGFP相比，融和蛋白在胞浆的表达相比胞核更强。更多还原

【Abstract】 Prostatic cancer is one of the most common cancer and death cause among men in the world especially in Euramerican countries. With the aging of population and development of examination,its incidence is increased year by year.In terms of the statistics of prostatic cancer incidence,it is ranked one of the fastest increased cancer in the ,21^st century in our country.In the past experiment,we found that gene PCaO5 was higher expressed in the expression database of the prostatic cancer tissue,its expression was 10 times higher than that in the common prostatic tissue.On the other hand, the expression of gene PCaO5 in the prostatic cancer tissue was higher than that in the other cancerous and common tissue.The result of Northern Blot originally indicated the upregulation of gene PCaO5 expression in the prostatic cancer. On the other hand, we did not find any report of this gene.According to PCaO5 sequence, a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized. RT-PCR of the total RNA extracted from LNCaP cell was performed, then abtained the objective PCaO5 gene segment. By digesting the eukaryotic expression vector pEGFP-C1 and objective PCaO5 gene segment with two types of restrictively nucleic acid inscribed enzyme Hind III and EcoR I, we obtained linearization pEGFP-C1 vector and objective PCaO5 gene segment, and joined them together, consequently gained recombined plasmid pEGFP-C1-PCaO5. The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the LNCaP cell by Lipofectamine 2000 and we observed their subcellular localization of pEGFP-C1-PCaO5 by the Laser scannning confocal microscope after 24 hour cell creep parcel.Some useful predicted information was obtained about gene function of PCa05, and PCaO5 is possibly a soluble protein regulated by oct-1 and revolved in regulating the gene expressing in celluar mitochondrion. The fusion protein was expressed in both nuclei and cytoplasm of LNCaP cell. However, compared with pure EGFP, the expression of更多还原