Development of ultra-small scale screening system using Tisgue engineering for human genomics based drug discovery

使用 Tisgue 工程开发超小规模筛选系统，用于基于人类基因组学的药物发现

基本信息

批准号：
12650790
负责人：
MATSUSHITA Taku
金额：
$ 2.62万
依托单位：
Sojo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

The whole genomic DNA sequence of human was almost determined in 2001, and the precise sequence will be determined in 2003. The important things for human genomics based drug discovery are construction of database about the relations between three-dimensional structure of human proteins presumed from DNA sequence and their functions, and development of efficient screening system for effective drugs among enormous genomics based compounds. Especially, the utilization of human normal cells for the screening system will be important, because it is found that drug metabolism of human is different from the other animal and information of drug designing is derived from human genomic DNA.In this research, three-dimensional culture (spheroid culture) technology originally developed by ourselves was applied to human normal hepatocytes to develop the efficient and ultra-small scale screening system for human genomics based drug discovery. Hepatocytes play an important role in drug metabolism in … More vivo. Then, we used human fetal hepatocytes supplied from US company based on the informed concent for research use as a human nomal hepatocyte.1. Serial proliferation of human fetal hepatocyte until 8 passages was achieved in serum-in and serum free medium.2. Morphology and function of human fetal hepatocytes were changed during proliferation. At over con fluent monolayer, the cells became epithelial hepatocytes and accumulated glycogens in the cells.3. Spheroid formation of human fetal hepatocytes was accelerated on the negatively charged surface of polystyrene dish, which was coated by poly-L-glutamic acid or poly-L-aspartic acid.4. Measurement method of cytochrome P450 activity in ultra-small scale was developed by using laser confocal microscopy. One spheroid made from 200-300 hepatocytes was enough for the measurement, which corresponded to 1/10,000 scale reduction compared to biochemical measurement.5. Cytochrome P450 (CYP1A1, CYP1A2, CYP2B1/2) activities of human hepatocyte/spheroids, which were main enzymes of drug metabolism, were 2.5〜3 times higher than those of usual monolayer culture. Less

2001年人类基因组DNA序列基本确定，2003年将确定精确序列。基于人类基因组学的药物发现的重要内容是建立关于从DNA推测的人类蛋白质的三维结构之间关系的数据库。特别是，利用人类正常细胞进行筛选系统将非常重要，因为人们发现人类的药物代谢与其他动物不同。和药物设计信息本研究将自主研发的三维培养（球体培养）技术应用于人类正常肝细胞，开发基于肝细胞基因组学的高效、超小规模筛选系统。然后，我们根据知情同意，使用美国公司提供的人胎儿肝细胞作为人正常肝细胞进行连续增殖，直至传代8次。 2．人胎儿肝细胞在细胞过度汇合时形态和功能发生变化，细胞内糖原不断积累。3．带负电荷的表面涂有聚L-谷氨酸或聚L-天冬氨酸的聚苯乙烯培养皿。 4．细胞色素P450活性的测定方法。使用激光共聚焦显微镜开发了一个由 200-300 个肝细胞制成的球体足以进行测量，与生化测量相比，相当于缩小了 1/10,000 的规模。5。 2）人肝细胞/球体的药物代谢主要酶活性为2.5〜3比通常的单层培养物高出几倍。