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1997 Fiscal Year Final Research Report Summary

Research for the in vitro reconstruction of hepatic tissues : an application for an artificial liver.

Research Project

Project/Area Number 08670260
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Experimental pathology
Research InstitutionSapporo Medical University School of Medicine

Principal Investigator

MITAKA Toshihiro  Department of Pathology, Cancer Research Institute, Sapporo Medical University Assistant Professor, がん研究所, 講師 (50231618)

Co-Investigator(Kenkyū-buntansha) MOCHIZUKI Yohichi  Department of Pathology, Cancer Research Institute, Sapporo Medical University P, がん研究所, 教授 (40045381)
Project Period (FY) 1996 – 1997
Keywordssmall hepatocytes / three-dimensional structure / stem cells / proliferation / differentiation / hepatic nonparenchymal cells / extracellular matrix
Research Abstract

A small hepatocyte can clonally proliferate for more than 3 months when the cells are cultured in DMEM supplemented with 10% FBS,10mM nicotinamide, 1mM ascorbic acid 2-phosphate, 10ng/mI EGF and 1% DMSO.The cell divides to form a colony and the number of the cells reaches to more than 100 cells within 20 days. With time in culture, cells with a large cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with a crystaline nucleoid, which are typical mature heatocytes. Immunoreactivity to Cx32 and well-developed bile canaliculus structures are often observed between cells. Thus, we suggest that small hepatocytes may be considered as "committed progenitor cells" that can further differentiate into mature hepatocytes.
We established a culture system in which, by adding 2% DMSO to the culture medium after the hepatocytes proliferate, the cells are able to recover differentiated functions such as albumin and transferrin secretion. Although expressions of connexin 32, Cx 26, and tryptophan 2,3-dioxygenase, thought to a highly differentiated function of mature hepatocytes, have never been maintained or re-induced in primary hepatocytes cultured for long period of time by using the conventional culture methods, those mPNA expressions were gradually restored with time after the addition of 2% DMSO and maintained for a month in this culture system.
We showed the role of the liver-enriched transcription factors in the transition during which proliferating hepatocytes become quiescent. We found that hepatic differentiation requires not only inhibition of DNA synthesis but also induction of appropriate transcription facors. Thus, expression of HNF3gamma, C/EBPalpha, and C/EBPbeta may be necessary for hepatocytes to acquire highly differentiated functions in addition to coexpression of certain amounts of transcripts of HNF1alpha, HNF1beta, HNF3alpha, HNF3beta, and HNF4 as well as suppression of C/EBPdelta.

  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Toshihiro Mitaka, et al.: "Growth and Maturation of Small Hepatocytes" Journal of Gastroenterology and Hepatology. (in press). (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 三高 俊広: "肝の再生:初代培養肝細胞研究から肝再生現象を考える" BIO Clinica. 13(印刷中). (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Toru Mizuguchi, et al.: "Alteration of expression of liver-enriched transcription factors in the transition between growth and differentiation" Journal of Cellular Physiology. 174. 273-284 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Takashi Kojima, et al.: "Different changes in expression and junction of connexin26 and connexin 32 during DNA synthesis and redifferentiation" Hepatology. 26. 585-597 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Takashi Kojima, et al.: "Effect of melatonin on proliferation,oxidative stress and Cx32 gap junction protein expression in primary cultures" Cell Structure and Function. 22. 347-356 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 三高 俊広: "肝臓の多能性幹細胞" Molecular Medicine. 34. 554-565 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Takashi Kojima, et al.: "Formation of actin filament networks in cultured rat hepatocytes treated with DMSO and glucagon" Cell Structure and Function. 22. 269-278 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Toru Mizuguchi, et al.: "Recovery of mRNA expression of tryptophan 2,3-oxygenase and serine dehydratase in the long-term cultures of primary rat" Journal of Biochemistry(Tokyo). 120. 511-517 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Takashi Kojima, et al.: "Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat" Experimental Cell Research. 223. 314-326 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Takashi Kojima, et al.: "Effects of oxygen radical scavengers on connexin 26 and 32 expression in primary cultures of adult rat hepatocytes" Carcinogenesis. 17. 537-544 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Toshihiro Mitaka, etal.: "Subculture of proliferating primary adult rat hepatocytes in medium supplemented with nicotinamide and EGF" In Vitro Cellular and Developmental Biology. 32. 469-477 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mitaka T,et al.: "Growth and maturation of small hepatocytes (review)" Journal of Gastroenterology and Hepatology. in press. (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Mizuguchic T,et al.: "Alteration of expression of liver-enriched transcription factors in the transition between growth and differentiation of primary cultured rat hepatocytes." Journal of Cellular Physiology. 174. 273-284 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kojima T,et al.: "Different changes in expression and junction of connexin 26 and connexin 32 during DNA synthesis and redifferentiation in primary rat hepatocytes using a DMSO culture system." Hepatology. 26. 585-597 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kojima T,et al.: "Effects of melatonin on proliferation, oxidative stress and Cx32 gap junction protein expression in primary cultures of adult rat hepatocytes." Cell Structure and Function. 22. 347-356 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kojima T,et al.: "Formation of actin filament networks in cultured rat hepatocytes treated with DMSO and glucagon." Cell Structure and Function. 22. 269-278 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Mizuguchic T,et al.: "Recovery of mRNA expression of tryptophan 2,3-oxygenase and serine dehydratase in the long-term cultures of primary rat hepatocytes." Journal of Biochemistry (Tokyo). 120. 511-517 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kojima T,et al.: "Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat hepatocytes." Experimental Cell Research. 222. 314-326 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kojima T,et al.: "Effects of oxygen radical scavengers on connexin 26 and 32 expression in primary cultures of adult rat hepatocytes." Carcinogenesis. 17. 537-544 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Mitaka T,et al.: "Subculture of proliferating primary adult rat hepatocytes in medium supplemented with nicotinamide and EGF." In Vitro Cellular and Developmental Biology, Animal. 32. 469-477 (1996)

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

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Published: 1999-03-16  

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