|Budget Amount *help
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
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
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