|Budget Amount *help
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
Use of primary cultures of hepatocytes in developing hybrid artificial liver is a topic of active research. However, primary cultures of hepatocytes remain viable for only a few weeks under normal cultivation conditions. By using adenovirus vector and introducing SV40 primary gene into rat and marmoset primary cultures of hepatocytes, I have suceeded in transformed conversion resulting in longer cultivation peroid, immortalization, and massive cultivation. By using the same technique, I have attempted to produce hybrid artificial liver by using human hepatocytes.
Methods and Results : 1.Adenovirus vecotr produced by recombination of E1A and E1B genes in human adenovirus with multiple deletion SV40 primary genes, was added to human primary cultures of hepatocytes by MOI (multiplicity of infection) and cultivated under 37ﾟC for two hours, then fixed in ethanol after 48 hours og additional cultivation, and immunostained by using antibody to SV40T antigen. Rate of T antigen positive was app
roximately 20% in MOI 100, 4% in MOI 10, less than 0.5% in MOI1 ; and these results were dependent on MOI.2.Adenovirus vector was introduced in human hepatocytes in MOI 100 and plated to 1x10^6/flask (25cm^2). After 3-4 weeks, approximately 100/colonies undergoing transformed conversion were produced from 10^6 cells. 3.Human hepatocytes introducing with primary SV40 genes were cultivated as a bulk for longer than 12 months and the cells mostly SV40T antigen positive, continued to reproduce favorably and attained immortalization. Also immunostaining with albumin was localization in cytosol of these cells.
Conclusion : By using adenovirus vector, efficient introduction of SV40 primary gene and transformed conversion was possible, resulting in massive cultivation and immortalization.
Discussion : Under the single layr cultivation method employed this time, hepatocyte function was found to deteriorated with time, by mesuring urea production and tyrosine amino transferase (TAT) activity. I plan to use third degree cultivation to improve this aspect in the future. Less