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

结直肠癌的Bradeion是本课题组发现的一个与细胞分裂和生长控制相关的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（“全部或无”类型表达式更改）;（3）没有由点突变、缺失、错义突变引起的任何氨基酸或结构变化，我们对一个基因进行了基础研究，诊断方法和在使用Bradeion的用于结肠直肠癌的基因治疗方法中的应用。建立一种简便的诊断方法（1）遗传筛查方法设计并合成Bradeion RT-PCR引物，利用培养的癌细胞和患者组织进行RT-PCR。作为这些研究的结果，很明显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。通过ELISA、免疫组织化学和Western blot对它们进行了评价，发现它们是有用的。进一步的研究是进行临床应用的测试条。Bradeion的生理功能分析（1）嵌合体小鼠的制作成功（专利申请中）。(2)我们成功地将Bradeion基因导入果蝇（日本专利2001-54759），通过双杂交方法发现Bradeion相关材料.通过控制Bradeion表达来开发基因治疗。(1)我们发现Bradeon特异性核酶导入培养的结直肠癌细胞后，细胞周期发生G2期阻滞。(2)为探索大肠癌的基因治疗方法，将携带Bradeon特异性核酶的大肠癌细胞接种于裸鼠。然后，具有Bradeion特异性核酶的结直肠癌细胞团不生长并且比阴性对照小鼠的结直肠癌细胞团小。

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.