| Project/Area Number |
06670532
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Gastroenterology
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| Research Institution | Shinshu University |
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Principal Investigator |
TANAKA Eiji Shinshu University Medical School Lecturer, 医学部, 講師 (50163506)
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| Project Period (FY) |
1994 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1995: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
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| Keywords | hepatitis C virus / gene cutter / gene therapy / ribozyme / asialoglycoprotein / polymer vector / gene cutter / アシアロ糖タンパク受容体 / Ribozyme / in situ hyhrid:zation |
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| Research Abstract |
HCV gene cutter consisted of ribozyme RNA and its carrier (asialoorosomucoid-poly-L-lysin ; ASOR-PLL). When a complex of biotinylated poly-C (RNA) and ASOR-PLL was injected intravenously to rat, biotinylated poly-C was detected only in hepatocyte, indicating specific delivery of RNA to hepatocyte by the carrier.
HCV RNA in cultured human hepatocyte was detected on sliced specimen by in-situ reverse transcription (in-situ RT). Further, nucleotide sequence specific cleavage of HCV RNA in hepatocyte by the ribozyme could be confirmed with the in-situ RT.
We made an animal model of HCV infection by transplanting HCV infected cultured human hepatocytes to the spleen of a rat. Ability of the gene cutter was tested using this model. Briefly, HCV gene cutter was infected intravenously to the rat. The spleen was removed 10 min. after the injection, and then the spleen which included transplanted hepatocytes were embedded in paraffin. In-situ RT was performed on a sliced specimen to detect the specific cleavage of HCV RNA by the ribozyme. RT of HCV RNA was not observed in the rat which was injected with the HCV gene cutter. On the other hand, RT of HCV RNA was observed in a control rat which was not injected with the gene cutter. These results indicate that our HCV gene cutter can cleave HCV RNA specifically in the animal model.
A polymer vector was studied to develop an effective system to introduce ribozyme into cytoplasm of hepatocyte where HCV replicates. Preliminary results indicated that oligo-DNA can be transported into the cytoplasm with the polymer vector.
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