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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

人类基因组DNA的全序列在2001年基本确定，精确的序列将在2003年确定。基于人类基因组学的药物发现的重要内容是建立基于DNA序列推测的人类蛋白质三维结构与其功能关系的数据库，以及开发基于基因组学的大量化合物中有效药物的高效筛选系统。特别是，利用人类正常细胞作为筛选系统将是重要的，因为发现人类的药物代谢与其他动物不同，药物设计的信息来源于人类基因组DNA。本研究将自主研发的三维培养（球体培养）技术应用于人正常肝细胞，开发基于人类基因组学的药物发现高效、超小型筛选系统。肝细胞在体内药物代谢中起着重要的作用。然后，我们使用美国公司提供的人类胎儿肝细胞作为人类正常肝细胞进行研究。人胎儿肝细胞在含血清和无血清培养基中连续增殖至8代。人胎肝细胞在增殖过程中形态和功能发生改变。在过浓缩的单层上，细胞变成上皮性肝细胞，并在细胞内积累糖原。3 .人胎肝细胞在带负电荷的聚苯乙烯培养皿表面涂覆聚l -谷氨酸或聚l -天冬氨酸，加速其球形形成。采用激光共聚焦显微技术，建立了超小尺度细胞色素P450活性的测定方法。一个由200-300个肝细胞制成的球体就足够测量了，与生化测量相比，减少了1/ 10000个尺度。细胞色素P450 （CYP1A1, CYP1A2, CYP2B1/2）活性是普通单层培养的2.5 ~ 3倍，是药物代谢的主要酶。少