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Semiquantification and localization of brain cytokine by in situ RT-PCR

Research Project

Project/Area Number 07680835
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Nerve anatomy/Neuropathology
Research InstitutionTokyo Metropolitan Institute for Neuroscience

Principal Investigator

KAWAZOE Yoko (1996)  Tokyo Metropolitan Institute for Neuroscience, 神経病理研究部門, 主任研究員 (60281705)

石原 好弘 (1995)  (財)東京都神経科学総合研究所, 神経病理研究部門, 副参事研究員 (50079711)

Co-Investigator(Kenkyū-buntansha) MATSUMOTO Yoh  Tokyo Metropolitan Institute for Neuroscience, 神経病理研究部門, 副参事研究員 (90173921)
申 台均  (財)東京都神経科学総合研究所, 神経病理研究部門, 研究員
Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
KeywordsExperimental autoimmune / in situ RT-PCR / Cytokine / encephalomyelitis
Research Abstract

In order to know the interaction between astrocytes and microglia during the course of experimental autoimmune encephalomyelitis (EAE), we tried to localize the cytokine mRNA in the central nervous system (CNS) by in situ RT-PCR after amplification of cytokine mRNA on tissue. Frozen or paraffin-embedded sections were pretreated with proteinase K and mRNA on sections was reverse transcribed into cDNA with the SuperScript Preamplification System. cDNA was then amplified using a TGF-beta1-specific primer pair in a thermal cycler by either the direct or indirect method. In the direct method, cDNA was amplified in the presence of DIG-dUDP and integrated DIG was detected by immunostaining for DIG.In the indirect method, amplification was performed using unlabeled nucleotides followed by in situ hybridization with DIG-labeled TGF-beta1-specific probe.
It was revealed that so-called " DNA repair artifact " in which DIG-dUDP was integrated into nicks of genomic DNA could not be eliminated in spite of several pretreatment procedures. As a result, all the nuclei on section were stained positively. On the other hand, such artifact was not observed in the indirect method. At the peak stage of EAE,many infiltrating inflammatory cells and microglia were stained positively. However, it was difficult to obtain stable staining partly because of poor fixation of amplified PCR products on tissue. In conclusion, optimal pretreatment procedures must be undertaken to obtain stable and specfic staining.

Report

(3 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • Research Products

    (24 results)

All Other

All Publications (24 results)

  • [Publications] Hirahara,H.et al.: "Long-term survival of caridac allografts in rats treated before and after surgery with monoclonal antibody to CD2" Transplantation. 59. 85-90 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Ikarashi,Y.et al.: "Recipient-derived T cells participate in autoimmune-like hepatic lesions induced by graft-versus-host reaction" Autoimmunity. 20. 121-127 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Shin,T.et al.: "The subarachnold space as a site for precursor T cell proliferation and effector T cell selection in experimental autoimmune encephalomyelitis." J.Neuroimmunol.56. 171-177 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Matsumoto,Y.et al.: "Characterization of CD4-CD8-TCRαβ+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis." Eur.J.lmmunol.26. 1328-1334 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tanuma,N.et al.: "Pretreatment with T cell receptor peptide using conventional immunizatior protocolodoes not induce effectiveprotection against autoimmune encephalomyelitis." Cell.lmmunol.168. 85-90 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tanuma,N.et al.: "Competitive PCR quantification of pro-and anti-inflammatory cytokine mRNA in the central nervous system during autoimmune encephalomyelitis" J.Neuroimmunol.(in press).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Hirahara, H.et al.: "Long-term survival of cardiac allografts in rats treated before and after surgery with monoclonal antibody to CD2" Transplantation. 59. 85-90 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Ikarashi, Y.et al.: "Recipient-derived T cells participate in autoimmune-like hepatic lesions induced by graft-versus-host reaction." Autoimmunity. 20. 121-127 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Shin, T.et al.: "The subarachnoid space as a site for precursor T cell proliferation and effector T cell selection in experimental autoimmune encephalomyelitis." J.Neuroimmunol.56. 171-177 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Matsumoto, Y.et al.: "Characterization of CD4-CD8- TCRalphabeta+T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis." Eur. J.Immunol.26. 1328-1334 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tanuma, N.et al.: "Pretreatment with T cell receptor peptide using conventional immunization protocol does not induce effective protection against autoimmune encephalomyelitis." Cell. Immunol.168. 85-90 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tanuma, N.et al.: "Competitive PCR quantification of pro- and anti- inflammatory cytokine mRNA in the central nervous system during autoimmune encephalomyelitis" J.Neuroimmunol.(in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Hirahara,H.et al.: "Long-term survival of cardiac allografts in rats treated before and after surgery with monoclonal antibody to CD2" Transplantation. 59. 85-90 (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] Ikarashi,Y.et al.: "Recipient-derived T cells participate in autoimmune-like hepatic lesions induced by graft-versus-host reaction." Autoimmunity. 20. 121-127 (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] Shin,T.et al.: "The subarachnoid space as a site for precursor T cell proliferation and effector T cell selection in experimental autoimmune encephalomyelitis." J.Neuroimmunol.56. 171-177 (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] Matsumoto,Y.et al.: "Characterization of CD4-DC8-TCRαβ+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis." Eur.J.Immunol.26. 1328-1334 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Tanuma,N.et al.: "Pretreatment with T cell receptor peptide using conventional immunization protocol does not induce effective protection against autoimmune encephalomyelti" Cell.Immunol.168. 85-90 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Tanuma,N.et al.: "Competitive PCR quantification of pro-and anti-inflammatory cytokine mRNA in the central nervous system during autoimmune encephalomyelitis" J.Neuroimmunol.(in press).

    • Related Report
      1996 Annual Research Report
  • [Publications] Hirahara, H. et al.: "Long-term survival of cardiac allografts in rats treated before and after surgery with monoclonal antibody to CD2" Transplantation. 59. 85-90 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Ikarashi, Y. et al.: "Recipient-derived T cells participate in autoimmune-like hepatic lesions induced by graft-versus-host reaction." Autoimmunity. 20. 121-127 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 田沼直之ら: "Competitive PCRによる自己免疫性脳脊髄炎におけるサイトカインmRNAの解析." 神経免疫学. 3. 48-49 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Matsumoto, Y. et al.: "Characterization of CD4-CD8-TCRαβ+T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis." Eur. J. Immunol.(in press).

    • Related Report
      1995 Annual Research Report
  • [Publications] Shin, T. et al.: "The subarachnoid space as a site for precursor I cell proliferation and effector T cell selection in experimental autoimmune encephalomyelitis." J. Neuroimmunol.56. 171-177 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Tanuma, N. et al.: "Pretreatment with T cell receptor peptide using conventional immunization protocol does not induce effective protection against autoimmune encephalomyelitis" Cell. Immunol.(in press).

    • Related Report
      1995 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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