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

人类基因组DNA全序列已于2001年基本确定，精确序列将于2003年确定。建立从DNA序列推测的人类蛋白质三维结构与其功能之间关系的数据库，以及开发高效的从大量基因组化合物中筛选有效药物的系统，是基于人类基因组学的药物发现的重要内容。尤其是利用人类正常细胞进行药物筛选具有重要意义，因为人类的药物代谢不同于其他动物，药物设计的信息来源于人类基因组DNA。本研究将自行开发的三维培养(椭球培养)技术应用于人类正常肝细胞，为基于人类基因组学的药物发现开发高效、超小型的筛选系统。肝细胞在…药物代谢中的重要作用更活生生的。然后，我们根据知情浓度将美国公司提供的人胎肝细胞用作人类正常肝细胞。结论：1.人胎肝细胞在有血清和无血清条件下连续扩增至8代。人胎肝细胞在增殖过程中形态和功能发生改变。在超过融合的单层时，细胞变为上皮性肝细胞，并在细胞内积聚糖原。聚L-谷氨酸或L-天冬氨酸均能促进人胎肝细胞在带负电的聚苯板表面形成球体。利用激光共聚焦显微镜建立了超微尺度细胞色素P450活性的测定方法。一个由200-300个肝细胞制成的球体就足以进行测量，与生化测量相比，这相当于规模缩小了1/10000。药物代谢的主要酶--人肝细胞/球体的细胞色素P450(CYP1A1、CYP1A2、CYP2B1/2)活性是普通单层培养的2.5~3倍。较少

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