Whole transcriptome analysis for ossification of vertebral ligament using RNA interference and microbeads array

2011 Fiscal Year Final Research Report

• Project/Area Number: 21591895
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Orthopaedic surgery
• Research Institution: University of Fukui
• Principal Investigator: UCHIDA Kenzo 福井大学, 医学部, 准教授 (60273009)
• Co-Investigator(Kenkyū-buntansha): NAKAJIMA Hideaki 福井大学, 医学部, 助教 (10397276)
• Project Period (FY): 2009 – 2011
• Keywords: 脊椎脊髄病学

Research Abstract

Histological, immunohistochemical, and real-time RT-PCR analyses of the expression of cell signaling and transcriptional factors in human ossified vertebral ligament. We analyzed the mRNA expression levels of signaling factors known to be involved in the ossification process in cultured ossified ligament cells subjected to cyclic tensile strain. Cyclic tensile strain was produced by Flexerce(R) FX-3000. The localization of these factors was examined in decalcified paraffin sections by immunohistochemistry. Controlled samples were harvested from non-ossified vertebral ligament of patients. Under resting conditions(no tensile strain), the mRNA levels ofβ-catenin, Runx2, Sox9 and Osteopontin in cultured ossified ligament cells were significantly higher than in the control cells. Application of cyclic tensile strain to ossified cells resulted in significant increases in mRNA expression levels ofβ-catenin, Runx2, Sox9, and Osteopontin at 24 hours. Hypertrophic chondrocytes present around the calcification front were immunopositive for Runx2 and Osteopontin. Immunoreactivity ofβ-catenin and Sox9 was strongly present in premature chondrocytes in the fibrocartilage area. Our results indicated that cyclic tensile strain applied to ossified vertebral ligament cells activated their ossification through a process mediated by theβ-catenin signaling pathway.

Research Products

• [Journal Article] Apoptosis of neurons and oligodendrocytes in the spinal cord of spinal hyperostotic mouse(twy/twy): possible pathomechanism of human cervical compressive myelopathy (2012)
  • Author(s): Uchida K, Nakajima H, Watanabe S, Yayama T, Guerrero AR, Inukai T, Hirai T, Sugita D, Johnson WE, Baba H
  • Journal Title: Eur Spine J Volume: 21 Pages: 490-497
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/21935678
  • Peer Reviewed
• [Journal Article] Ossification process involving the human thoracic ligamentum flavum : role of transcription factors. (2012)
  • Author(s): Uchida K, Yayama T, Cai HX, Nakajima H, Sugita D, Guerrero AR, Kobayashi S, Yoshida A, Chen KB, Baba H
  • Journal Title: Arthritis Res Ther Volume: 13 Pages: R144
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/21914169
  • Peer Reviewed
• [Journal Article] Initiation and progression of ossification of the posterior longitudinal ligament of the cervical spine in the hereditary spinal hyperostotic mouse(twy/twy) (2012)
  • Author(s): Uchida K, Yayama T, Sugita D, Nakajima H, Rodriguez Guerrero A, Watanabe S, Roberts S, Johnson WE, Baba H
  • Journal Title: Eur Spine J Volume: 21 Pages: 149-155
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/21850419
  • Peer Reviewed
• [Journal Article] High-mobility group box-1 and its receptors contribute to proinflammatory response in the acute phase of spinal cord injury in rats (2011)
  • Author(s): Chen KB, Uchida K, Nakajima H, Yayama T, Hirai T, Rodriguez Guerrero A, Kobayashi S, Ma WY, Liu SY, Zhu P, Baba H
  • Journal Title: Spine(Phila Pa 1976) Volume: 36 Pages: 2122-2129
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/21343866
  • Peer Reviewed
• [Journal Article] Tumor necrosis factor-αantagonist reduces apoptosis of neurons and oligodendroglia in rat spinal cord injury (2011)
  • Author(s): Chen KB, Uchida K, Nakajima H, Yayama T, Hirai T, Watanabe S, Guerrero AR, Kobayashi S, Ma WY, Liu SY, Baba H
  • Journal Title: Spine(Phila Pa 1976) Volume: 36 Pages: 1350-1358
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/21224756
  • Peer Reviewed
• [Journal Article] Microarray analysis of expression of cell death-associated genes in rat spinal cord cells exposed to cyclic tensile stresses in vitro (2010)
  • Author(s): Uchida K, Nakajima H, Hirai T, Yayama T, Chen KB, Kobayashi S, Roberts S, Johnson WE, Baba H
  • Journal Title: BMC Neurosci Volume: 11 Pages: 84
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/20663127
  • Peer Reviewed
• [Journal Article] Targeted retrograde gene delivery of brain-derived neurotrophic factor suppresses apoptosis of neurons and oligodendroglia after spinal cord injury in rats (2010)
  • Author(s): Nakajima H, Uchida K, Yayama T, Kobayashi S, Guerrero AR, Furukawa S, Baba H
  • Journal Title: Spine(Phila Pa 1976) Volume: 35 Pages: 497-504
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/20190624
  • Peer Reviewed
• [Journal Article] Tumor necrosis factor-alpha and its receptors contribute to apoptosis of oligodendrocytes in the spinal cord of spinal hyperostotic mouse(twy/twy) sustaining chronic mechanical compression (2009)
  • Author(s): Inukai T, Uchida K, Nakajima H, Yayama T, Kobayashi S, Mwaka ES, Guerrero AR, Baba H
  • Journal Title: Spine(Phila Pa 1976) Volume: 34 Pages: 2848-2857
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/19949368
  • Peer Reviewed
• [Journal Article] Calcium pyrophosphate dehydrate crystal deposition in the ligamentum flavum of the cervical spine : histopathological and immunohistochemical findings (2009)
  • Author(s): Mwaka ES, Yayama T, Uchida K, Kobayashi S, Kokubo Y, Nakajima H, Sato R, Orwotho NT, Baba H
  • Journal Title: Clin Exp Rheumatol Volume: 27 Pages: 430-438
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/19604435
  • Peer Reviewed
• [Journal Article] Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice
  • Author(s): Guerrero AR, Uchida K, Nakajima H, Watanabe S, Nakamura M, Johnson WE, Baba H
  • Journal Title: J Neuroinflammation Volume: (in press)
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/22369693
  • Peer Reviewed
• [Journal Article] Transplantation of Mesenchymal Stem Cells Promotes an Alternative Pathway of Macrophage Activation and Functional Recovery after Spinal Cord Injury
  • Author(s): Nakajima H, Uchida K, Guerrero AR, Watanabe S, Sugita D, Takeura N, Yoshida A, Long G, Wright KT, Johnson WE, Baba H
  • Journal Title: J Neurotrauma Volume: (in press)
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/22233298
  • Peer Reviewed
• [Journal Article] Cyclic Tensile Strain Facilitates the Ossification of Ligamentum Flavum Throughβ-Catenin Signaling Pathway
  • Author(s): Uchida K, Yayama T, Cai CH, Nakajima H, Sugita D, Guerrero AR, Yoshida A, Baba H
  • Journal Title: In Vitro Analysis Spine(Phila Pa 1976)(in press) Volume: (in press)
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/22158061
  • Peer Reviewed
• [Presentation] Cycli ctensile stress upregulates the expression ofβ-catenin signaling in cultured cells harvested from human ossified ligamentum flavum (2012)
  • Author(s): 彌山峰史,内田研造,中嶋秀明,杉田大輔,馬場久敏
  • Organizer: Annual Meeting of the Orthopaedic Research Society
  • Place of Presentation: San Francisco
  • Year and Date: 20120206-07
• [Presentation] Cyclic tensile stressが胸椎黄色靱帯の骨化形成に与える影響-内軟骨性骨化過程におけるWntβ-catenin signalingの関与- (2011)
  • Author(s): 彌山峰史,内田研造,中嶋秀明,杉田大輔,馬場久敏
  • Organizer: 第26回日本整形外科学会基礎学術集会
  • Place of Presentation: 前橋
  • Year and Date: 2011-10-20
• [Presentation] 脊柱靭帯骨化における骨化前線部の軟骨細胞分化の特徴. (2011)
  • Author(s): 彌山峰史,内田研造,杉田大輔,中嶋秀明,小久保安朗,渡邉修司,吉田藍,竹浦直人,馬場久敏
  • Organizer: 第30回日本運動器移植・再生医学研究会
  • Place of Presentation: 福岡
  • Year and Date: 2011-09-25
• [Presentation] 培養骨化靱帯細胞における転写因子の発現とtensile stressの影響 (2011)
  • Author(s): 彌山峰史,内田研造,中嶋秀明,小久保安朗,杉田大輔, Cai Hong-xin,吉田藍,馬場久敏
  • Organizer: 第40回日本脊椎脊髄病学会
  • Year and Date: 2011-04-22
• [Presentation] Cell death-associated genes expression patterns using microarray analysis in rat cultured spinal cord cells exposed to cyclic tensile stresses in vitro (2010)
  • Author(s): K. Uchida, H. Nakajima, T. Hirai, T. Yayama, D. Sugita, S. Kobayashi, H. Baba
  • Organizer: 7^<th> Combined Meeting of ORS
  • Place of Presentation: KYOTO
  • Year and Date: 2010-10-17
• [Presentation] Topographic expression of transcriptional factors in the human ossification of ligamentum flavum in thoracic spine (2010)
  • Author(s): Yayama T, Uchida K, Cai HX, Kobayashi S, Nakajima H, Sato R, Sugita D, Alexander Guerrero, Chen KB, Baba H
  • Organizer: 7^<th> Combined Meeting of ORS
  • Place of Presentation: KYOTO
  • Year and Date: 2010-10-17
• [Presentation] 培養骨化靱帯細胞における機械的ストレスと骨形成誘導因子 (2010)
  • Author(s): 彌山峰史、内田研造、Cai HX、中嶋秀明、小久保安朗、小林茂、杉田大輔、馬場久敏
  • Organizer: 第29回日本運動器移植・再生医学研究会
  • Place of Presentation: 京都
  • Year and Date: 2010-09-25