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1998 Fiscal Year Final Research Report Summary

Studies on proliferating mechanism of hepatitis C virus using cultured human liver cell lines by radialflowtype-bioreactor

Research Project

Project/Area Number 09480257
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Biomedical engineering/Biological material science
Research InstitutionJIKEI UNIVERSITY SCHOOL OF MEDICINE

Principal Investigator

NAGAMORI Seishi  JIKEI UNIVERSITY SCHOOL OF MEDICINE,FUCULTY OF MEDICINE,ASSOCIATE PROFESSOR, 医学部, 助教授 (60119831)

Co-Investigator(Kenkyū-buntansha) MATSUURA Yashiharu  NATIONAL INSTITUTE OF INFECTIOUS DISEASE,LABOCHIEF, ウイルス第2部, 室長 (50157252)
MIYAMURA Tatsuo  NATIONAL INSTITUTE OF INFECTIOUS DISEASE,DIRECTOR, ウイルス第2部, 部長 (90100099)
MATSUURA Tomokazu  JIKEI UNIVERSITY SCHOOL OF MEDICINE,FUCULTY OF MEDICINE,INVESTIGATER, 医学部, 助手 (30199749)
HASUMURA Satoshi  JIKEI UNIVERSITY SCHOOL OF MEDICINE,FACULTY OF MEDICINE,LECTURER, 医学部, 講師 (30189518)
Project Period (FY) 1997 – 1998
Keywordsartificial bio-liver / hepatitis C virus / replication-deficient recombinant / FLC4 / hepatocellular carcinoma
Research Abstract

In order to carry out a construction of infectious clones to infect into the artificial bio-liver, we constructed a full length cDNA clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood sample was shown to be infectious to both a recipient and chimpanzees. Then we constructed a recombinant baculovirus containing the full length cDNA of HCV by using a charomid system. By immunoprecipitation analysis, properly processed HCV proteins were expressed in cells infected with the recombinant baculovirus.
Cell lines were then assessed for their suitability in artificial models of HCV infection and replication. We constructed a replication-deficient recombinant adenovirus expressing bacteriophage T7 RNA polymerase under the control of CAG promoter (AdexCAT7). A high level of T7 RNA polymerase was detectable. Cells infected with AdexCAT7 were then transfected with plasmids carrying (i) the T7 promoter, the 5' untranslated region (UTR) of encephalomyocarditis virus and … More a luciferase (pT7EMCVLuc), or (ii) the T7 promoter, the 5'UTR of HCV and a luciferase gene (pT7HCVLuc). Most of the cell lines examined supported a higher expression of luciferase by transfection with pT7EMCVLuc than with p7F7HCVLuc. However, one cell line, FLC4, derived from a human hepatocellular carcinoma exhibited very high reporter gene expression with pT7HCVLuc. In this cell line, transfection with RNA synthesized in vitro from pT7HCVLuc induced a higher level of reporter gene expression than RNA from pT7EMCVLuc. It may be due to robust stability of the HCV RNA minigene transcription process in FLC4 owing to some as yet unknown factor. In monolayer culture, FLC4 cells showed continuous secretion of HCV RNA when experiments were carried out using plasma taken from a chronic Hepatitis C patient Taken together, these findings persuaded us to select FLC4 cells as the most suitable for further studies in artificial models of HCV infection and replication. Stable high-density cultivation in bioreactor environments was successfully carried out for more than 60 days, and furthermore, proliferation rates could be controlled by cultivating at lower temperatures.
Using this artificial liver model, it was possible to confirm by RT-PCR the secretion of HCV RNA in response to Hepatitis C infected plasma HCV RNA tested positive for the first two days after infection, but tests were negative thereafter. Unfortunately, infection of the culture medium could not be confirmed.
We are presently continuing studies using the infectious clones we have developed as well as those obtained from the NIH USA which have been shown to infect chimpanzees. Less

  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] 永森 静志: "新しい長期三次元高密度大量培養法による人工肝補助装置-ラジアルフロー型バイオリアクターの機能評価-" 肝臓. 38. 268-282 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 永森 静志: "特集(人工肝臓への道-肝細胞とバイオクアクターの集歩)に寄せて" 組織培養工学. 23. 280-283 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 永森 静志: "人工肝補助装置の開発" カレントテラピー. 16・11. 2106-2110 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 永森 静志: "人工肝補助装置を用いた肝解毒機能の検討" 肝臓. 39. 507-508 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Seishi Nagamori: "Massive caltare of human liver cancer cells ra newly developed radial flow bioreanfor sgstem" In Vitro. Cell.Dev.Biol.34. 109-115 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Seishi Nagamori: "Full-length complenertary DNA of hyafifis C Virus. genome from an infectious blood Sample" Hepatology. 27. 2. 621-627 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 永森 静志: "キーワードを読む : 消化器 人工臓器・人工肝(人工肝補助装置)" 医学書院, 168 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 永森 静志: "肝臓病の最前線 1997" 中外医学社, 409 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] NAGAMORI Seishi et al.: "Artificial liver support system using three dimensional culture by newly developed radial flow boireactor" ACTA HEPATOLOGICA JAPONICA. 38. 268-282 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] NAGAMORI Seishi et al.: "Development of artificial liver (Progress in bioreactor and liver cells)" THE TISSUE CULTURE ENGINEERING. 23. 280-283 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] NAGAMORI Seishi et al.: "Development of liver support system" CURRENT THERAPY. 16-11. 2106-2110 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] NAGAMORI Seishi et al.: "Studies on mechanisms of drug metabolism by using artificial liver" ACTA HEPATOLOGICA JAPONICA. 39. 507-508 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] NAGAMORI Seishi et al.: "Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system" IN VITRO CELL DEV.BIOL. 34. 109-115 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] NAGAMORI Seishi et al.: "Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample" HEPATOLOGY. 27-2. 621-627 (1998)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-12-08  

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