|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1997 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Current therapy of chronic hepatitis C with a 6 months course of alpha interferon shows limited effectiveness for patients with chronic hepatitis C. In particular, more effective therapy is needed to treat patients who have a high serum level of HCV RNA. We previously reported that combination of interferon and cyclosporin A (CsA) was more effective than interferon alone (4th international meeting on hepatitis C virus and related virus). Antiviral effect of CsA on HCV has not been fully elucidated. In the present study, we examined whether CsA has an antiviral effect on HCV using cultured cell line.
We used IMY-N9 cell which was hybrid cell line from human hepatocytes and HepG2 cells to propagate HCV. Infection was performed by 1 hour incubation of IMY-N9 cell suspension with an inoculum in serum free medium. Following exposure, cells were washed twice with Williams medium and seeded on 48 well plate. CsA was dissolved with dimethyl sulfoxide (DMSO). Final concentration of CsA and DMSO
were 100ng/ml, 1000ng/ml, 10000ng/ml and 0.01%. We confirmed that CsA and DMSO were not toxic for IMY-N9 cell using alamar blue assay. On day 1, medium was changed and added each concentration of CsA (CsA group), DMSO alone (vehicle control group) or none (control group). Cells were harvested on day 2, 4, 6 and 8 and stored -80C until use. HCV RNA in cultured cells were quantitatively determined by RTD-PCR based on Taq Man chemistry. Immediately after seeding, HCV RNA in cultured cells was 2x103 copies/well. On day 2 and 4, HCV RNA in cultured cells decreased to 4x102 copies/well in the presence or absense of CsA or DMSO. On day 6, HCV RNA in control and vehicle control groups increased to 1.3-1.5x104 copies/well. In CsA groups (100ng/ml, 1000ng/ml and 10000ng/ml), HCV RNA were reduced to 80%, 40% and 20% of control level, respectively. On day 8, HCV RNA returned to 3-5x102 copies/well in all groups. Propagation of HCV seems to be inhibited by CsA. Further study is needed to clarify the action of CsA. Less