【作者】 武广恒；

【导师】 张家颖；

【作者基本信息】 吉林大学 ， 免疫学， 2007， 硕士

【摘要】 目前,乳腺癌作为女性群体中常见的恶性肿瘤,其发病率呈逐年上升趋势。深入探讨细胞因子的变化与乳腺癌发生的相关性,从分子水平研究乳腺癌的发病机理和预防手段,正日益引起人们的关注。干扰素是一类多功能细胞因子,对治疗包括肿瘤在内的许多疾病都具有一定的疗效。IFN-γ基因+874位点的单核苷酸多态性与大肠癌以及乙型肝炎等疾病的相关性已见报道,但与乳腺癌的关系国内尚未见到有关资料。本研究从94例乳腺癌患者及96例正常女性全血中提取基因组DNA,根据Genbank数据库中公布的IFN-γ基因序列,采用primer 5.0软件设计出3条特异性引物,进行PCR扩增。PCR产物的特异性采用酶切鉴定及测序证实。按照其单核苷酸的多态性,将PCR产物电泳后分为AA、AT和TT等三种不同基因型,将TT基因型目的片断连接入pGEM-T载体,构建重组质粒,进行测序分析。比较病例组与对照组之间的基因型频率及等位基因频率,进行统计学分析。结果显示,乳腺癌病例组IFN-γ基因+874位点TT基因型频率以及T等位基因频率均显著高于对照组,提示该位点TT基因型可能与乳腺癌的发生有关。本实验探讨了IFN-γ基因+874位点的单核苷酸多态性分布,分析其与乳腺癌的相关性,为临床治疗和预防提供了新的理论参考。更多还原

【Abstract】 As the rapid development of genetic engineering and the Immunologic technology and the constant intensification of mankind understanding to the pathogenesis of breast cancer,cell factors such as interferon,etc. become one of the focuses studied in tumour biology gradually, immune gene therapy has already become the important component in treatment of breast cancer, the application prospect of it is extremely broad..The emergence of breast cancer is a complicated course based on the activation of oncogenes or the inactivation of tumor suppressor genes, these hereditary foundation and other carcinogenesis factors and environmental factors act on and demonstrate cancer together. The common oncogenes of breast cancer are as follows, c-erbB-2, ras gene, c-myc gene,etc;the TSG of breast cancer mainly includes p53 gene, FHIT gene, BRCA1 gene,etc. With the further development of molecular biology and immunology, it becomes possible to know the pathogenesyof tumour from essence.B cells, monocytes, natural killer cells and dendritic cells can produce interferon by the stimulation of numerous antigen such as viruses bacterium, nucleotides and some other micromolecules.Since the study of influenza finding the interfering phenomenon in 1957, the research to interferon is deepened constantly, The study in genetic engineering and gene therapy of interferon attracted extensive attention.The sources of interferons are different in different cell type, property of different inductor and the condition of induction .IFN can generally be divided into IFN–α、IFN-βand IFN-γ. IFN–α、IFN -βis called the Model I IFN jointly; IFN -γis called the ModelⅡIFN or immune IFN.Interferon is a kind of multifunctional cytokine, a high biological active protein synthesized and secreted by somatic cells, showing biological functions on against viral infections and tumor growth, modulating and enhancing host immune. Therefore, Interferon takes central roles on anti-virus and anti-tumor immunity in immunological defense. IFN I induced by the virus, microorganisms and their products is responsible for anti-virus and anti-tumor. IFNⅡgenerated from lymphocyte to which mutagens or specific antigens are given can effectively activate macrophagesand NK cells, maintain B-cell proliferation and differentiation, induce the expression of MHC class I antigens and immune regulation effects, kill a variety of tumor cells.Gene expression of IFN is modulated by many of transcription factors, cytokines, regulatory element, such as interleukins, IFN regulatory factor (IRF), and cytokinins. DNA sequencing showed that interferon is a type of ancient biological protection factor. The experiments show that interferon can control tumor growth and inhibit tumor metastasis in vivo or in vitro. Interferon can inhibit tumor growth through by anti-proliferation, promoting apoptosis and inhibiting tumor angiogenesis and immune regulation. Recent studies found that interferon may have a direct cytotoxic effect on tumors. IFN can bind to specific receptors on the cell membrane and induce target cell to produce specific proteins, and trigger a cascade of signal amplification process to deliver signals into nucleus for regulating the expression of many genes and leading to physiological responses. IFN plays a role in immune regulation through enhancing phagocytic activity of macrophages and special target cell cytotoxicity of lymphocytes. With development of molecular biology and DNA recombination technology, the mechanism of anti-tumor of IFN will be cleared and be a help for clinic tumor therapeutic strategies.Our experiments focus on single nucleotide polymorphism of +874 sites in breast cancer IFN-γgene to study the relativity corresponding with initiation of breast carcinoma. Single nucleotide polymorphism (SNP), as the third generation genetic marker, is the most common variety in human genome and has been used frequently in studies of genecology and disease-related genes. SNP is a single base variety in certain sites of chromosome DNA. In present, most of SNP detective methods base on PCR technology. Two strategies of SNP research were used as followed: First, to detect unknown SNP. The number of genetic map marker has been increased to find the target gene marker; Second, to screen known SNP for SNP typing in samples. The second one has been used in the experiment in merit of rich site and reliability. The genomic DNA were extracted from blood samples of 96 normal women and 94 cases of female breast cancer patients in the experiment. The PCR was performed using designed primers. The gene type was identified byelectrophoresis and SNP was analyzed. The results showed: TT gene frequency appeared to be different between normal group and breast cancer group; Allele frequency showed a significant distinguish between normal group and breast cancer group, TT gene frequency and T allele frequency was higher in breast cancer group than in normal group.The single nucleotide polymorphism of colorectal Cancer IFN-γgene in +874 sites has been reported. However, the relationship to breast cancer has not been reported. Our results showed that TT gene type of IFN-γ+874 site might have a relationship with generation of breast cancer.Our research provided a helpful reference to breast cancer on early prevention, accurate diagnosis and prompt treatment. With developing of cell biology and molecular biology, breast cancer will be known deeply on genetic study and pathogenesis. The studies focus on genetic reason and relative cytokines will help in early diagnosis and prognosis of breast cancer.更多还原