【作者】 姜飞；

【导师】 刘小林；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 前列腺癌是男性特有的肿瘤,也是全球男性常见的恶性肿瘤疾病,死亡率高居第二位。目前,前列腺癌的发病率呈逐年上升趋势。前列腺癌发展过程漫长且隐匿,临床标本难以获得。因此,常用模拟前列腺癌发展的细胞或动物模型开展前列腺癌基础研究。利用前列腺分泌蛋白94（PSP94）启动子建立的基因敲入前列腺癌小鼠模型（KIMAP）,显示与人类相近的肿瘤特征。本研究以KIMAP前列腺癌小鼠模型为研究对象,主要包括以下内容:（1）利用基因芯片对不同生长时期（20周龄和60周龄）的小鼠进行差异表达基因筛选,筛选高上调表达未知功能基因;并结合反转录聚合酶链式反应（RT-PCR）和实时定量PCR方法对筛选出来的高上调表达未知功能基因进行验证;（2）利用RT-PCR技术对筛选出的一个小鼠高上调表达未知功能基因编码区序列进行克隆,并利用生物信息学方法分析其蛋白质结构;（3）构建该未知功能基因的原核表达载体,利用亲和层析技术纯化重组蛋白,Western-blot技术检测重组蛋白;（4）利用纯化后的重组蛋白免疫家兔,制备该未知功能基因的多克隆抗体;（5）利用获得的多克隆抗体进行该基因功能研究。获得的主要研究结果如下:（1）小鼠前列腺癌高上调表达基因的筛选。本研究成功进行前列腺癌模型小鼠基因芯片检测,结果表明在20周龄的模型小鼠中发现上调基因491个（大于等于正常小鼠的2倍）,60周龄的模型小鼠中发现上调基因442个（大于等于正常小鼠的2倍）。本研究选择上调10倍以上的基因进行分析。前列腺组织表达上调10倍以上的基因有72个,其中67个为已知功能基因,5个为未知功能基因。对5个未知功能基因进行RT-PCR和实时荧光PCR方法验证,结果与基因芯片结果相一致。（2）小鼠未知功能基因TRIM59的编码区克隆及生物信息学分析。利用RT-PCR技术克隆小鼠TRIM59基因的编码区序列,其编码区长度为1212 bp。将小鼠TRIM59（NM₀25863）与人TRIM59（NM₁73084）、鸡TRIM59（NM₁73084）的核苷酸和氨基酸进行同源性分析,核苷酸序列同源性分别为85 %和72 %;氨基酸序列同源性分别为86 %和75 %。对TRIM59的氨基酸结构、潜在功能位点等进行分析,预测结果表明TRIM59第10⁵9位氨基酸为RING结构域、第92¹34位氨基酸为B-box结构域、第329³48位氨基酸为跨膜结构域;1³28位氨基酸残基编码膜外蛋白,349⁴03位氨基酸残基编码膜内蛋白;通过磷酸化位点分析发现该蛋白具有潜在的蛋白磷酸化修饰。（3）小鼠TRIM59基因的原核表达。本研究特别选取TRIM59编码第277～403位的C末端126位氨基酸（cDNA序列的961¹338位碱基）作为抗原区进行克隆。将TRIM59基因的部分编码区插入pGEX-2T载体中成功构建原核表达载体;结果在大肠杆菌BL21中通过IPTG（异丙基-β-D-硫代半乳糖苷）诱导,成功表达带谷胱甘肽转移酶（GST）标签的45 kDa左右的TRIM59重组蛋白,且经过Western Blotting检测该蛋白表达正确。通过收集纯化后的GST-TRIM59重组蛋白,为制备TRIM59多克隆抗体奠定重要的基础。（4）TRIM59兔抗鼠多克隆抗体的制备与抗体的应用。利用之前制备纯化的GST-TRIM59重组蛋白,免疫家兔成功地制备兔抗鼠TRIM59多克隆抗体,经鉴定具有良好的免疫学活性,能够用于试验研究。利用获得的TRIM59抗体进行荧光免疫试验发现TRIM59主要定位于NIH3T3的细胞胞浆执行其生物功能。Western blotting印迹和免疫组化染色显示TRIM59在前列腺癌组织中的表达明显高于正常组织,验证小鼠前列腺癌芯片的关于TRIM59的结果,也为TRIM59作为前列腺癌诊断标志物提供参考。（5）TRIM59的RING结构域功能研究。试验结果表明,突变后以及缺失RING结构域的TRIM59在体内外试验中,均无法与泛素形成多聚肽链的结构,只有正常TRIM59 （与E2结合酶UbcH5a或UbcH5c共同作用）具有与泛素形成多聚肽链结构的能力,说明RING结构域是TRIM59具有泛素连接酶活性的重要功能域,半胱氨酸（Cys）对RING结构的生物学功能的保证具有重要作用。本研究通过对前列腺癌模型小鼠基因芯片结果的筛选,研究与前列腺癌相关未知基因的功能,为寻找新的前列腺癌治疗靶点和诊断标志物奠定一定的理论基础。更多还原

【Abstract】 Prostate cancer （PCa） is the most frequently diagnosed cancer for men. PCa has become the second leading cause of death in male malignant carcinomas. More significantly, the incidence of PCa has been increasing in recent years among Chinese. Simulated development of PCa cells and animal models are commonly used in preclinical research on PCa studies. A knock-in mouse adenocarcinoma prostate model （KIMAP） at a locus of PSP94 （prostate secretory protein of 94 amino acids） gene was therefore established. The KIMAP showed kinetics of tumor development which is close to human conditions. Considering the reasons mentioned above, the concerned study was carried out based on the following components: （1） Microarray used to screen the differential highly expressed genes with unknown function in prostate between wild type mice and KIMAP prostate cancer mouse model （by 20th week and 60th week）. The RT-PCR analysis and RT-qPCR analysis were used to validate the result of screening. （2） The coding sequence of mouse gene with unknown function was cloned by RT-PCR from total RNA of the mouse prostate. The structural domain, putative signal sequences, transmembrane domain, and secondary & tertiary structure of protein were predicted by bioinformatics. （3） The prokaryotic expression vector was constructed and expressed in prokaryotic cells. The purified protein was obtained via affinity chromatography. And the recombination protein was detected by Western blotting. （4） The purified fusion protein was used as immunogen for immunizing the rabbits. （5） The Gene functions were studied with polyclonal antibody. Results listed as following:（1） Screening the expression of KIMAP mouse model related genes by microarray. The results indicated that 491 genes with their expression up-regulated more than 2-fold from 20th week KIMAP mouse comparing to the wild type mouse; 442 genes up-regulated more than 2-fold from 60th week KIMAP mouse comparing to the wild type mouse. There were 72 genes which up-regulated more than 10-fold in KIMAP mouse model comparing to the wild type mouse. The function of 67 genes were known in highly expressed genes （72 genes）, and the function of 5 genes （72 genes） were unknown. The genes of unknown function were verified by RT-PCR results and RT-qPCR results.（2） The whole coding sequence （1212 bp） of TRIM59 gene was gained, which encoded a polypeptide of 403 amino acids. The homologies of cDNA nucleotide sequence of TRIM59 （NM₀25863） with the counterparts of human （NM₁73084） and chick （NM₀01031320） were 85% and 72% respectively, and the homologies of peptide sequence were 86% and 75% respectively. TRIM59 was a mono transmembrane protein and the transmembrane domain was located at 329-348 peptides. The TRIM59 protein included 3 functional domains of RING （10-60 peptides）, B-box （92-134 peptides） and transmembrane （329-348 peptides）.（3） We got the fragment of mouse TRIM59 with the coding region （961-1338 bp） by RT-PCR. Then the target fragment was cloned into Prokaryotic expression vector pGEX-2T using the recombinant DNA technique and it was confirmed by DNA sequence analysis and restriction enzyme digestion. After induced by IPTG, the fusion protein was expressed in E.coli BL21 at 45 kDa. The fusion protein was purified and collected via affinity chromatography（4） We immunized rabbits with purified fusion protein （GST-TRIM59）. The rabbits were injected with purified fusion protein as immunogen; we collected the immune sera of those rabbits. The sera separated from the blood and the antibody （IgG） separated from the immune sera were purified by Protein A agrose. Those antibodies titers and specifies detected by ELISA and Western blotting. Indirect immunofluorescence analysis showed that TRIM59 located in the cytoplasma of NIH3T3, indicating as the possible main function place for TRIM59. TRIM59 highly expressed in prostate cancer cells which was detected by Western blotting and IHC.（5） Our study demonstrated that E3 ubiquitin ligase activity was a novel property of TRIM59. It was found that TRIM59 underwent self-ubiquitylation in vitro when combined with the E2 enzyme UbcH5a and UbcH5c.The ubiquitylation was dependent on its RING finger domain. Further evidences showed that TRIM59 could also be self-ubiquitylated in vivo. Our data showed that Cys was an important amino-acid residue of RING domain to keep E3 ubiquitin ligase.In this research, prostate cancer-related genes of unknown function have been studied via microarray screening and functional study within prostate cancer mouse model. It lays the theoretical foundation for identifying new therapeutic targets and diagnostic markers for prostate cancer.更多还原