Bradeion, the gene expressed in colorectal cancer specifically --its functional analysis and technical development of clinical application--

结直肠癌的Bradeion是本课课组发现的与细胞分裂和生长控制有关的septin家族基因。Bradeion基因可以被认为是结直肠癌的标志物，原因如下：(1)细胞类型的选择性；(2)特异性（“全或无”型表达变化）；(3)在表型改变过程中，没有任何由点突变、缺失、错义突变等引起的氨基酸和结构变化。我们开展了一种（基因）诊断方法的基础研究，并应用于Bradeion对结直肠癌的基因治疗方法。(1)基因筛选方法bradeion RT-PCR引物设计合成，利用培养的癌细胞和患者组织进行RT-PCR。这些研究的结果表明，Bradeion RT-PCR诊断方法是有用的。(2)试纸条法的应用表达、提取、纯化Bradeion融合蛋白，制备特异性单克隆抗体。采用ELISA、免疫组织化学和Western blot对其进行评价。进一步的临床应用研究正在进行中。Bradeion的生理功能分析(1)我们成功制造了一只嵌合体小鼠（在专利申请期间）。(2)成功地将Bradeion基因引入日本果蝇。专利2001-54759)用双杂化方法发现与Bradeion相关的材料。控制Bradeion表达的基因治疗进展。(1)我们发现，在培养的结直肠癌细胞中引入bradeion特异性核酶可以在细胞周期中发生G2阻滞。(2)为开发结直肠癌的基因治疗方法，将转染bradion特异性核酶的结直肠癌细胞接种于裸鼠。携带Bradeion特异性核酶的结直肠癌细胞团块不再生长，且体积小于阴性对照小鼠。

Bradeion, for colorectal cancer, was discovered to be a septin family gene related to cell division and growth control by our research group. The Bradeion gene can be considered as a marker for colorectal cancer in the following reasons:(1) selectivity of cell types; (2) specificity ("all or none" type expression changes); (3) neither any amino acid nor structural changes caused by point mutations, deletions, miss-sense mutation, etc. in the course of phenotype change.We carried out the basic study of a (gene) diagnostic method and application to a gene therapy method using Bradeion for the colorectal cancer.1. Development of a simple diagnostic method(1) Genetic screening methodBradeion RT-PCR primers were designed and synthesized, and RT-PCR using cultivated cancer cells and patient tissues. As the results of these studies, it became clear that a Bradeion RT-PCR diagnostic method is useful.(2) Application for test strip methodAs Bradeion fusion protein was expressed, extracted and purified, specific monoclonal antibodies were made. They were evaluated by ELISA, immunohistochemistry and Western blot, and found useful. Further studies are proceeded for clinical application to a test strip.2. Physiological function analysis of Bradeion(1) We succeeded in making of a chimera mouse (during a patent application).(2) We succeeded in introduction of Bradeion gene to fruit fly (Jap. Patent 2001-54759) to discover Bradeion -relating materials by Two-hybrid method.3. Development of the gene therapy by a control of Bradeion expression.(1) We discovered that G2 arrest in a cell cycle was occurred by Bradeion-specific ribozymes introduction to a cultivated colorectal cancer cells.(2) To develop gene therapy method of colorectal cancer, colorectal cancer cells in which Bradeion-specific ribozymes were transfected were inoculated in nude mice. Then the masses of colorectal cancer cells with Bradeion -specific ribozymes did not grow and smaller than those of negative control mice.