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Molecular analysis for the mechanisms of endoplate sclerosis using laser microdissection technique

Research Project

Project/Area Number 18591623
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Orthopaedic surgery
Research InstitutionTokyo Medical and Dental University

Principal Investigator

KOGA Daisuke  Tokyo Medical and Dental University, 医学部附属病院, Assistant (60422482)

Co-Investigator(Kenkyū-buntansha) ASOU Yoshinori  Tokyo Medical and dental University, 医学部附属病院, Junior Associate Professor (50345279)
SHIMOJI Takashi  (財)癌研究会, The genome center of Japanese foundation of cancer research, Research fellow (40370150)
TAKEDA Shu  Tokyo Medical and dental University, 大学院・医歯学総合研究科, Associate Professor (30376727)
SHINOMIYA Kenichi  Tokyo Medical and dental University, 大学院・医歯学総合研究科, Professor (20111594)
Project Period (FY) 2006 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥3,920,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,100,000 (Direct Cost: ¥2,100,000)
Keywordsdegeneration of intervertebral disc / animal model / laser microdissection / マイクロダイゼクション / 網羅的検索 / 分子生物学
Research Abstract

The pathogenesis of intervertebral disc degeneration is poorly understood, but is known to be associated with a variety of cellular and biochemical changes. Vertebral endplate changes and bone marrow changes visible in magnetic resonance imaging (MRI), are closely associated with dipc degeneration. To investigate molecular mechanisms underlying the process of endplate degeneration, we tried to apply laser capture microdissetion combined with microarray analysis to vertebral endplate.
Although we have succeeded in extract total RNA from vertebral endplate, its quantity has not been enough for microarray analysis. Thus, we could not help abandon carry out the microarray analysis. However, we could acquire many skills for the analysis of hard tissues including bone and cartilage. Besides, we have developed novel disc degeneration model for rat tail.
We have already developed continuous weight bearing disc degeneration model for mouse tail. In this model, degradation of IVD was observed with … More in two weeks after operation. Using this model, we tested if weight loading, a most common cause of intervertebral disc (IVD) degeneration, induces Runx2 expression, which may cause chondrocyte hypertrophy. Indeed, weight loading of the IVD by compression for a day significantly increased Runx2 expression. Thus, we next examined the expression of Runx2 with other disc degradation markers in canine IVD, The expression of RUNX2, type-X collagen and MMP-13 mRNA in young intact and degenerated IVD were examined by semi-quantitative RT-PCR analysis. The localization of Runx2 and type-X collagen protein in control and degenerated IVD were examined by imunohistochemistly. The expression of Runx2 transcript and protein, in combination with type-X collagen and MMP-13, was enhanced in degenerated IVDs of the dog.
Next, we have investigated the relationship between expression patterns of Runx2 and the degree of intervertebral disc degeneration in canine NP cells through comparing the expression patterns to MMP-13 expression, a disc degeneration marker, and to MRI findings. Canine disc specimens were obtained from chondrodystrophoid dogs, including Dachshund and Beagle. RACE method indicated the canine Runx2 cDNA showed over 97% conservation with human RUNX2 and 95% with mouse. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that mRNA levels of Runx2 and MMP-13 were similarly increased in degenerated NP and herniated specimens. Immunohistochemical analysis indicated that T2/CSF ratio in MRI images was significantly correlated with Runx2 protein expression rate in various age beagle disc samples. Thus, our findings suggest Runx2 is a novel marker for disc degeneration.
As mentioned above, intervertebral disc degeneration induced by mechanical compression is an important issue in spinal disorder research. In this study, the biomechanical aspect of the rat tail model was also investigated. An external loading device equipped with super-elastic TiNi springs was developed to apply a precise load to the rat tail. By using this device, rat tail discs were subjected to compressive stress of 0.5 or 1.0 MPa for 2 weeks. Discs in the sham group received an attachment of the device but no loading. After the experimental period, first the intact tail with peripheral tissues (PT) such as tendon and skin and then the retrieved disc without PT were subjected to a uniaxial tension-compression test ; biomechanical characteristics such as range of motion (ROM), neutral zone (NZ), and hysteresis loss (HL) were evaluated. Furthermore, the loadbearing contribution of PT in the intact tail was estimated by comparing the load-displacement curves obtained by the mechanical tests performed with and without PT.The experimental findings revealed that the continuous compressive stress induced reduction in disc thickness. The intact tail demonstrated decreases in ROM and NZ as well as increases in HL.On the other hand, the retrieved disc demonstrated increases in ROM, NZ, and HL.Further, a significant increase in the load-bearing contribution of PT was indicated. These findings suggest that the load-bearing capacity of the disc was seriously deteriorated by the application of compressive stress of 0.5 or 1.0MPa for 2 weeks.
Lastly, we have developed novel LCM technique applied for hard tissues, such as articular cartilage. Using our techniques, we have been investigating the mechanisms of cartilage degeneration. Less

Report

(3 results)
  • 2007 Annual Research Report   Final Research Report Summary
  • 2006 Annual Research Report
  • Research Products

    (16 results)

All 2009 2008 2007 2006 Other

All Journal Article (8 results) (of which Peer Reviewed: 3 results) Presentation (8 results)

  • [Journal Article] TNF-α-Induced NF-kB Signaling Reverses Age-Related Declines in VEGF Induction and Angiogenic Activity in Intervertebral Disc Tissues2009

    • Author(s)
      麻生義則, ほか
    • Journal Title

      Journal of Orthopaedics Research 27

      Pages: 229-235

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Effects of compressive loading on biomechanical properties of disc and peripheral tissue in a rat tail model.2009

    • Author(s)
      麻生義則, ほか
    • Journal Title

      European Spine Journal 18

      Pages: 1595-1603

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Effects of compressive loading on biomechanical properties of disc and peripheral tissue in a rat tail model2009

    • Author(s)
      Asou Y, et. al.
    • Journal Title

      Eur Spine J. 18(11)

      Pages: 1595-603

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] TNF-α-induced NF-κB signaling reverses age-related declines in VEGF induction and angiogenic activity in intervertebral disc tissues2009

    • Author(s)
      Asou Y, et. al.
    • Journal Title

      J Orthop.Res. 27(2)

      Pages: 229-35

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Enhanced type X collagen expression in the extruded nucleus pulposus of the chondrodystrophoid dog.2008

    • Author(s)
      麻生義則、四宮謙一, ほか
    • Journal Title

      Journal of Veterinary Medical Science 70

      Pages: 37-42

    • NAID

      130000447240

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Enhanced type X collagen expression in the extruded nucleus pulposus of the chondrodystrophoid dog2008

    • Author(s)
      Asou Y, Shinomiya K, et. al.
    • Journal Title

      J.Vet.Med.Sci. 70(1)

      Pages: 37-42

    • NAID

      130000447240

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Runx2 expression correlates with the degree of disc degeneration : a comparison between Magnetic Resonance Imaging and Runx2 expression.

    • Author(s)
      古賀大介、麻生義則、四宮謙一, ほか
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Runx2 expression in the canine disc : cooperative expression with Matrix Metalloproteinase-13 in herniated discs

    • Author(s)
      Koga D, Shinomiya K., Asou Y, et. al.
    • Journal Title

      (in submit)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Leptin regulation of articular chondrocyte is independent of mechanical stress or sympathetic nerve stimuraltion.2009

    • Author(s)
      古賀、麻生、竹田、四宮, ほか
    • Organizer
      米国骨代謝学会
    • Place of Presentation
      米国
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Leptin regulation of articular chondrocyte is independent of mechanical stress or sympathetic nerve stimuraltion2009

    • Organizer
      Annual meeting of American society for bone and mineral research
    • Place of Presentation
      US
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] 変性椎間板におけるMMP-13と転写因子Runx2の発現解析2007

    • Author(s)
      麻生義則、竹田秀、四宮謙一, ほか
    • Organizer
      日本整形外科基礎学術集会
    • Place of Presentation
      国内
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Runx2 expression in the canine disc : cooperative expression with Matrix Metalloproteinase-13 in herniated discs2007

    • Author(s)
      Asou Y, Takeda S, et. al.
    • Organizer
      Annual orthopaedic research meeting of the Japanese orthopaedic
    • Place of Presentation
      Japan
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] 犬の椎間板逸脱症罹患症例の逸脱椎間板物質におけるRunx2の発現について2006

    • Author(s)
      麻生義則、竹田秀、四宮謙一, ほか
    • Organizer
      日本獣医畜産大学学術交流会
    • Place of Presentation
      国内
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Runx2 Expression correlates with Magnetic Resonance Imaging intervertebral disc degeneration.2006

    • Author(s)
      麻生義則、竹田秀、四宮謙一, ほか
    • Organizer
      骨代謝学会
    • Place of Presentation
      米国
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Runx2 Expression correlates with Magnetic Resonance Imaging intervertebral disc degeneration2006

    • Author(s)
      Asou Y, Shinomiya K, et. al.
    • Organizer
      Annual meeting of American society for bone and mineral research
    • Place of Presentation
      US
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Degeneration of intervertebral discs by compression2006

    • Author(s)
      Asou Y, et. al.
    • Organizer
      JSME Conference on Frontiers in Bioengineering
    • Place of Presentation
      Japan
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary

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Published: 2006-04-01   Modified: 2016-04-21  

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