【作者】 胡海燕；

【导师】 刘贤锡；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 鸟氨酸脱羧酶(Ornithine decarboxylase, ODC)(EC 4.1.1.17)是多胺代谢过程中的第一个限速酶。多胺广泛存在于真核生物细胞中,含有多个碱性的氨基,能与核酸分子相互结合,并促进DNA的生物合成,RNA的转录以及蛋白质的翻译,起到稳定细胞结构、增强核酸和蛋白质的合成的作用,为细胞的增殖提供物质基础从而促进细胞的增殖。凡生长旺盛的组织,如胚胎、再生肝等组织,细胞中多胺的含量均较高。高特异性的多胺合成抑制剂可以干扰非洲爪蟾卵母细胞的激素诱导的ODC活性,随后抑制其减数分裂及排卵。多胺的代谢与肿瘤的发生也有着密切的关系。早期的研究发现,病毒诱导原代成纤维细胞转化时,腐胺和精脒的含量增多。多胺在维持肿瘤细胞恶性表型中也起重要作用,抑制多胺合成可抑制肿瘤细胞增殖并诱导其分化及凋亡,引起肿瘤恶性表型逆转。由于多胺为ODC的产物,因此ODC对细胞的正常发育及异常增殖也起重要作用。 ODC及多胺水平升高几乎是所有种类的癌细胞的特征。用强力的肿瘤促进剂刺激上皮细胞后的早期变化之一就是ODC活性迅速、显著、一过性的诱导升高。ODC的合成受多种因素的影响,激素、生长因子、肿瘤刺激因子、病毒以及癌基因均能诱导ODC的合成,从而使细胞内的多胺水平增加。ODC基因表达可在转录、mRNA的降解、mRNA的翻译、酶蛋白的降解几个水平上进行调控。ODC基因被认为是一个立早基因,其中包含的应答元件可与几种反式作用因子结合,包括cAMP应答元件,胰岛素应答元件及几个Sp1结合位点等。ODC基因的诱导还对多种原癌基因的信号传导具有重要作用,如ras,v-src和myc基因被激活后对细胞的转化过程中常伴随着ODC基因的表达和多胺含量的增加。正常细胞中生长因子对ODC的调节是快速而短暂的,而肿瘤刺激因子、病毒及癌基因对ODC的作用是持续的。这些事实表明细胞内的ODC过度表达伎多胺生物合成增多,可能是细胞过度增殖,最终导致肿瘤发生的重要原因之一。更多还原

【Abstract】 Ornithine decarboxylase(ODC, 4.1.1.17) is the first rate-limiting enzyme in polyamine biosynthesis. The polyamines are naturally occurring aliphatic polycations found in almost all living cells. It contains putrescine, spermidine, and spermine, which play an important role in cell proliferation, differentiation, and transformation. They are positively charged at neutral pH and the charge is distributed along the length of the molecule. This facilitates their interaction with anionic molecules such as DNA and RNA. Polyamines can interact with membranes, which generally leads to an decrease in the fluidity of the membrane. This has important consequences for the movement of proteins and lipids within the membrane. In the early 1970s, the late Diane Russell first observed that patients with malignant disease excreted higher amounts of polyamines in their urine than normal individuals. As with other tumours, polyamine content of colorectal cancers is increased when compared to the equivalent normal tissue.In addition to changes in polyamine content, Over-expression of ODC activity is a well-recognized feature of many cancers. As the first and rate-limiting enzyme, ODC is the most extensively studied enzyme of polyamine metabolism. The ODC protein is 50kDa as an monomer and about lOOkDa when forming the active dimmer. ODC synthesis is dramatically induced by different growth stimuli, such as hormones, growth factors,carcinogens, viruses and oncogens. The regulation can occur at the levels of transcription, translation and protein degradation. The alteration in enzyme can occur very quickly and change polyamine level in the end. ODC gene is considered as an immediate early gene, and contains response elements for several trans-acting factors, including a CAMP response element, a possible insulin response element and several Sp1 binding sites. In addition, ODC gene expression is tightly linked to transformation by activated ras, v-src and myc. So, recently, ODC gene is postulated as an oncogene that is essential for cell transformation. As to colorectal cancer ODC is found in very limited amounts in quiescent cell and its activity is found to increase significantly in colon adenocarcinoma and prostate tissue compares to normal tissue from the same patients. Colorectal carcinoma is a major health problem in the west and become more and more popular in the east. Colorectal adenoma increases sharply in the six decade of life and occur in more than half the population by the ninth decade of life, but colorectal cancer develops in only small part of the population. With the improvement of surgical techniques, more effective adjuvant therapy and earlier diagnosis, mortality has been decreasing over the last decade. However, older individuals still need to improve the survival rate by molecular approaches.The same as other cancers, colorectal cancer is a mutifactorial disease with about 75%of cases being sporadic and only 25% being linked to other conditions such as chronic inflammatory disease, and family history of colonrectal neoplasia. Many risk factors have been identified. In addition to advancing age and conditions mentioned above, the diet such as rich in fat, low fiber and calcium, tobacco use and sedentary lifestyle have play an important role in the disease initiation and progression. Laboratory testing and screening has the potential favorable effect in patients outcome and survival rate. Studies have been directed onidentifying markers for later development of colorectal cancer. Polyamine content as well as ODC activity has been found to be increased significantly in adenocarcinoma tissue compared to paired nomal tissue. Colorectal cancer tissue had approximately 4 times the polyamine content of the colonic mucosa from patients with non-malignant disease and Serum polyamine levels were also increased in patients with colorectal carcinoma. It was shown that wild-type APC decreased the levels of ODC RNA and the ODC promoter activity. These research results suggested that ODC might be used as an identifying marker for later development of additional primary tumors.The present study examined the ODC gene expression in human colorectal carcinoma and normal colon mucous. ODC mRNA was extracted from human colon cancer tissues and ODC mRNA were detected by RT-PCR. The ODC cDNA was inserted into expression vector and the ODC gene expression vector pQE-ODC has been established. The vector has a 6-His tag which made the expression product ODC protein of about 95% pure was used as a good immunogenic agent to immunize the BALB/c mouse. Monoclonal anti-ODC antibody is produced by cell hybrids between hypoxanithine phosphoriboxyltransferase deficient myeloma cells and spleen cells of immunized mouse. The antibody is a IgG2a type.ELISA and western blotting showed that the mAb could combine the ODC protein. Staining the colorectal tissues with ODC mAb showed significant difference between normal and tumor mucous. The deep brown color staining could be found mostly in gland cells of the tumor while there are no stains or weak stains in stoma cells. The results suggested that there was high concentration of ODC in tumor cell cytoplasm. Moderately differentiated carcinoma cells demonstrate most cells and deepest staining than others. More samples should be tested. RT-PCR showed that the ODC mRNA was much higher in colon tumor tissues than in normal tissues. These changes didnot correlate with gender, histological grade or Duck’ s stage, which confirmed that ODC was both early and late events in the colon carcinoma sequence in all patients. The continuously change in ODC content play an important role in the diagnosis of human colorectal carcinoma. Part One: Cloning, Expression and Purify of Human ODC Protein METHODS:1. According to the sequence of the human ornithine decarboxylase gene, a pair of primers specific for amplifying the complete ODC cDNA were designed and synthesized.2. Total RNA were extracted from normal and cancer tissues, respectively. The method of RNA extraction was similar to the Trizol RNA extraction protocol. ODC cDNA was synthesized by RT-PCR3. The product of PCR was inserted into a T-A clone vector pMD18-T.4. The recombinant was transformed into E. coli. DH5 a , Then the plasmid was extracted, purified and digested with BamH I and Sal I . The insert fragment was collected.5. The expression vector pQE30 were digested by BamH I and Sal I . The large fragment was linked with the target DNA fragment. The recombinant plasmid was identified arid sequenced.6. The positive recombinant was transformed into an expression strain of E. col i. M15 and the bacteria was induced to express ODC protein by IPTG.7. The protein was purified by Ni-NTA affinity chromatography and tested by SDS-PAGE.RESULTS AND CONCLUSION:1. Using the designed primers, the complete encoding sequence of ODC gene was amplified from human ODC total RNA.2. A expression plasmid contain ODC cDNA was constructed by ligating the expression vector pQE30 with ODC cDNA.3. The protein was purified by Ni-NTA affinity chromatographyPart Two: Preparation of Monoclonal Antibody Against Human ODC METHODS:1. The BALB/C female mouses were immunized with the prepared ODC protein direct admistrating into the spleen and were reimmunized every two weeks by intraperitoneally.2. The monoclonal antibodies against human ODC were prepared with hybridoma technique.3. The antibody in the supernatant of cell clones were tested by ELISA and the positive hybridoma cells were recloned four times in HAT medium by limiting dilution.4. The purified ODC protein were separated by standard SDS-PAGE and tested by Western blot.5. The chromosom of the cell strains were tested. RESULT AND CONCLUSION:1. 4 strains of hybridoma cells that could secret monoclonal antibodies stably were obtained.2. The chromosome number of hybridoma cells was between 100-110 that is about the total number of the two mother cells. There were metacentric and submetacentric chromosomes in hybridoma cell.3. ELISA showed that the subtype of the McAb was IgG2a with high specificity and affinity.Part Three: Using of The Obtained McAb METHOD:1. Test the ODC expression in colon carcinoma by Immunohistochemical staining using the prepared anti-ODC McAb.2. Test the ODC expression in colon carcinoma by Western blot using the prepared anti-ODC McAb.RESULT AND CONCLUTION:1. Immunohistochemical analysis shows that density staining in deep browncolor in most of the colon carcinoma tissues while few faint stainingin the normal tissue.2. The same result can be seen in Western blot.更多还原