软骨调节素在成人退变椎间盘中的表达
|
摘要
[研究背景]我们实验室前期的研究结果显示,在青少年特发性脊柱侧凸(AIS)患者侧凸顶点终板软骨凸凹侧蛋白质组存在显著差异,这些存在表达差异的蛋白很可能与椎间盘的不对称发育有关。同时由于脊柱侧凸可导致椎间盘承受异常的应力,AIS患者椎间盘在成熟度和退变程度上与同龄正常人群存在显著差异,这些存在表达差异的蛋白也很可能参与了椎间盘退变的病理过程。软骨调节素(chondromodulin-Ⅰ, ChM-Ⅰ)是仅在凸侧表达的关键蛋白之一。其主要作用在与促进软骨生长,维持软骨及所在组织的无血管活性,并与骨关节炎、心脏瓣膜病以及骨、软骨肿瘤的发病有关。有关ChM-Ⅰ在椎间盘退变过程中的作用还未见深入报道。
[目的]
1.利用分子生物学及免疫组织化学方法研究ChM-Ⅰ在成人退变椎间盘细胞中的表达情况。
2.观察碱性成纤维细胞生长因子(bFGF)对椎间盘细胞ChM-Ⅰ表达的影响。
3.观察ChM-Ⅰ在不同退变程度椎间盘组织中的表达情况。
4.对ChM-Ⅰ在椎间盘退变过程中可能发挥的作用作初步探讨。
[方法]
1.取3例因腰椎间盘退变性疾病而需行后路椎间融合患者的椎间盘组织分别进行髓核及纤维环细胞培养。取部分原代细胞利用RT-PCR和Western blot研究ChM-ⅠmRNA和蛋白在成人退变椎间盘细胞中的表达情况。
2.利用不同浓度(0ng/ml, 1ng/ml和10ng/ml) bFGF刺激椎间盘细胞,利用real-time RT-PCR和Western-Blot研究不同浓度bFGF对髓核细胞和纤维环细胞ChM-ⅠmRNA和蛋白表达的影响。
3.收集因腰椎间盘退变性疾病而行手术切除的椎间盘组织标本28例,根据MRI表现退变程度为Ⅲ-Ⅴ级,作为退变组。收集因脊柱肿瘤行手术治疗时切除的椎间盘标本6例,退变程度Ⅰ级,作为对照组。利用免疫组织化学方法研究ChM-Ⅰ在不同退变程度椎间盘组织中的表达情况。
[结果]
1. RT-PCR和Western blot结果显示ChM-I在椎间盘髓核细胞及纤维环细胞中均有表达,两者间在表达程度上无显著差异。
2. bFGF可明显抑制ChM-I mRNA和蛋白在髓核细胞及纤维环细胞中的表达,且呈剂量依赖性(P<0.05)。
3. ChM-I在对照组(无退变椎间盘组织)表达量很低,而在椎间盘发生退变后其表达量明显升高,ChM-I在不同退变程度椎间盘组织中的表达存在显著差异(P<0.05)。
[结论]
1.首次利用RT-PCR和Western blot方法证实髓核及纤维环细胞可表达ChM-I。
2.髓核细胞和纤维环细胞中ChM-I的表达受bFGF的影响。
3.椎间盘发生退变后ChM-I表达明显升高,提示其可能作为一种防御机制参与了椎间盘退变的病理过程。
Background Previous study of our laboratory showed that there were different protein expression levels between the convex and concave sides of the intervertebral discs endplate cartilage in AIS patients. The key proteins which express on the convex or concave side exclusively may involved in the progress of the AIS. Recent study showed that morphologic disc degeneration was accelerated about 20 years in scoliosis versus physiological ageing. The result indicated that the key proteins may also involved in the progress of intervertebral disc degeneration. Chondromodulin-I (ChM-I) is one of the key proteins which express on the convex side exclusively. ChM-I can stimulate the proliferation of chondrocytes and maintain the avascularity and anti-angiogenic properties of normal cartilage. It is also associated with the progress of osteoarthritis, valvular heart diseases and tumor. Yet the role of ChM-I in intervertebral disc degeneration is still unkown.
Objectives
1. To investigate the expression of ChM-I in the intervertebral disc cells.
2. To investigate the effect of bFGF on the expression of ChM-I in the intervertebral disc cells.
3. To examine the expression of ChM-I and to correlate its expression with the degree of disc degeneration.
4. To discus the role of ChM-I in the disc degeneration.
Methods
1. Three IVDs obtained from patients in the treatment of disc degenerative disease were used for cell culture. ChM-I expression in IVD cells was examined by using RT-PCR and Western blot.
2. The effect of bFGF on the expression of ChM-I was assessed by real-time PCR and Western blot respectively.
3. Twenty-eight human IVD tissues were obtained from patients in the surgical treatment of disc degenerative disease at different stage of degeneration according to MRI.6 IVD tissues removed from patients with metastatic spinal tumor were used as normal control. The expression of ChM-I determined by immunohistochemical analyses was correlated with MRI degeneration grade.
Results
1. RT-PCR and Western blot examination showed that ChM-I expressed in both adult degenerative anulus fibrosus cells and nucleus pulposus cells.
2. The mRNA and protein expression of ChM-I were both down-regulated by administration of bFGF with dose-dependent way(P<0.05)
3. Immunohistochemical analyses showed the number of ChM-I-expression cells in human IVD tissues was significantly elevated in advanced stages of IVD(grade III, IV, V) degeneration compared with those in normal control(P<0.05)
Conclusions
1. ChM-I mRNA and protein are expressed in IVD cells.
2. The expression of ChM-I is down-regulated by administration of bFGF.
3. The expression of ChM-I in discs with advanced stage of degeneration is elevated compared with those with nondegeneration which indicate that the expression of ChM-I is upregulated through a defense mechanism against the degenerative process of intervertebral disc.
[目的]
1.利用分子生物学及免疫组织化学方法研究ChM-Ⅰ在成人退变椎间盘细胞中的表达情况。
2.观察碱性成纤维细胞生长因子(bFGF)对椎间盘细胞ChM-Ⅰ表达的影响。
3.观察ChM-Ⅰ在不同退变程度椎间盘组织中的表达情况。
4.对ChM-Ⅰ在椎间盘退变过程中可能发挥的作用作初步探讨。
[方法]
1.取3例因腰椎间盘退变性疾病而需行后路椎间融合患者的椎间盘组织分别进行髓核及纤维环细胞培养。取部分原代细胞利用RT-PCR和Western blot研究ChM-ⅠmRNA和蛋白在成人退变椎间盘细胞中的表达情况。
2.利用不同浓度(0ng/ml, 1ng/ml和10ng/ml) bFGF刺激椎间盘细胞,利用real-time RT-PCR和Western-Blot研究不同浓度bFGF对髓核细胞和纤维环细胞ChM-ⅠmRNA和蛋白表达的影响。
3.收集因腰椎间盘退变性疾病而行手术切除的椎间盘组织标本28例,根据MRI表现退变程度为Ⅲ-Ⅴ级,作为退变组。收集因脊柱肿瘤行手术治疗时切除的椎间盘标本6例,退变程度Ⅰ级,作为对照组。利用免疫组织化学方法研究ChM-Ⅰ在不同退变程度椎间盘组织中的表达情况。
[结果]
1. RT-PCR和Western blot结果显示ChM-I在椎间盘髓核细胞及纤维环细胞中均有表达,两者间在表达程度上无显著差异。
2. bFGF可明显抑制ChM-I mRNA和蛋白在髓核细胞及纤维环细胞中的表达,且呈剂量依赖性(P<0.05)。
3. ChM-I在对照组(无退变椎间盘组织)表达量很低,而在椎间盘发生退变后其表达量明显升高,ChM-I在不同退变程度椎间盘组织中的表达存在显著差异(P<0.05)。
[结论]
1.首次利用RT-PCR和Western blot方法证实髓核及纤维环细胞可表达ChM-I。
2.髓核细胞和纤维环细胞中ChM-I的表达受bFGF的影响。
3.椎间盘发生退变后ChM-I表达明显升高,提示其可能作为一种防御机制参与了椎间盘退变的病理过程。
Background Previous study of our laboratory showed that there were different protein expression levels between the convex and concave sides of the intervertebral discs endplate cartilage in AIS patients. The key proteins which express on the convex or concave side exclusively may involved in the progress of the AIS. Recent study showed that morphologic disc degeneration was accelerated about 20 years in scoliosis versus physiological ageing. The result indicated that the key proteins may also involved in the progress of intervertebral disc degeneration. Chondromodulin-I (ChM-I) is one of the key proteins which express on the convex side exclusively. ChM-I can stimulate the proliferation of chondrocytes and maintain the avascularity and anti-angiogenic properties of normal cartilage. It is also associated with the progress of osteoarthritis, valvular heart diseases and tumor. Yet the role of ChM-I in intervertebral disc degeneration is still unkown.
Objectives
1. To investigate the expression of ChM-I in the intervertebral disc cells.
2. To investigate the effect of bFGF on the expression of ChM-I in the intervertebral disc cells.
3. To examine the expression of ChM-I and to correlate its expression with the degree of disc degeneration.
4. To discus the role of ChM-I in the disc degeneration.
Methods
1. Three IVDs obtained from patients in the treatment of disc degenerative disease were used for cell culture. ChM-I expression in IVD cells was examined by using RT-PCR and Western blot.
2. The effect of bFGF on the expression of ChM-I was assessed by real-time PCR and Western blot respectively.
3. Twenty-eight human IVD tissues were obtained from patients in the surgical treatment of disc degenerative disease at different stage of degeneration according to MRI.6 IVD tissues removed from patients with metastatic spinal tumor were used as normal control. The expression of ChM-I determined by immunohistochemical analyses was correlated with MRI degeneration grade.
Results
1. RT-PCR and Western blot examination showed that ChM-I expressed in both adult degenerative anulus fibrosus cells and nucleus pulposus cells.
2. The mRNA and protein expression of ChM-I were both down-regulated by administration of bFGF with dose-dependent way(P<0.05)
3. Immunohistochemical analyses showed the number of ChM-I-expression cells in human IVD tissues was significantly elevated in advanced stages of IVD(grade III, IV, V) degeneration compared with those in normal control(P<0.05)
Conclusions
1. ChM-I mRNA and protein are expressed in IVD cells.
2. The expression of ChM-I is down-regulated by administration of bFGF.
3. The expression of ChM-I in discs with advanced stage of degeneration is elevated compared with those with nondegeneration which indicate that the expression of ChM-I is upregulated through a defense mechanism against the degenerative process of intervertebral disc.
引文
[1]Mori Y, Hiraki Y, Shukunami C, et al. Stimulation of osteoblast proliferation by the cartilage-derived growth promoting factors chondromodulin-I and-II[J]. FEBS Lett,1997,406(3):310-314.
[2]Funaki H, Sawaguchi S, Yaoeda K, et al. Expression and localization of angiogenic inhibitory factor, chondromodulin-I, in adult rat eye[J]. Invest Ophthalmol Vis Sci,2001,42(6):1193-1200.
[3]Yoshioka M, Yuasa S, Matsumura K, et al. Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis[J]. Nat Med,2006,12(10):1151-1159.
[4]Hayami T, Funaki H, Yaoeda K, et al. Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis[J]. J Rheumatol,2003,30(10):2207-2217.
[5]Mera H, Kawashima H, Yoshizawa T, et al. Chondromodulin-1 directly suppresses growth of human cancer cells[J]. BMC Cancer,2009,9:166.
[6]Freemont A J, Peacock T E, Goupille P, et al. Nerve ingrowth into diseased intervertebral disc in chronic back pain[J]. Lancet,1997,350(9072):178-181.
[7]Peng B, Hao J, Hou S, et al. Possible pathogenesis of painful intervertebral disc degeneration[J]. Spine (Phila Pa 1976),2006,31(5):560-566.
[8]Hiraki Y, Tanaka H, Inoue H, et al. Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes[J]. Biochem Biophys Res Commun,1991,175(3):971-977.
[9]Inoue H, Kondo J, Koike T, et al. Identification of an autocrine chondrocyte colony-stimulating factor:chondromodulin-I stimulates the colony formation of growth plate chondrocytes in agarose culture[J]. Biochem Biophys Res Commun,1997,241(2):395-400.
[10]Hiraki Y, Kono T, Sato M, et al. Inhibition of DNA synthesis and tube morphogenesis of cultured vascular endothelial cells by chondromodulin-I[J]. FEBS Lett,1997,415(3):321-324.
[11]Yanagihara I, Yamagata M, Sakai N, et al. Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells[J]. J Bone Miner Res,2000,15(3):421-429.
[12]Hiraki Y, Tanaka H, Inoue H, et al. Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes[J]. Biochem Biophys Res Commun,1991,175(3):971-977.
[13]Azizan A, Holaday N, Neame P J. Post-translational processing of bovine chondromodulin-I[J]. J Biol Chem,2001,276(26):23632-23638.
[14]Aoyama T, Okamoto T, Nagayama S, et al. Methylation in the core-promoter region of the chondromodulin-I gene determines the cell-specific expression by regulating the binding of transcriptional activator Sp3[J]. J Biol Chem,2004,279(27):28789-28797.
[15]Aoyama T, Okamoto T, Kohno Y, et al. Cell-specific epigenetic regulation of ChM-I gene expression:crosstalk between DNA methylation and histone acetylation[J]. Biochem Biophys Res Commun,2008,365(1):124-130.
[16]Shukunami C, Iyama K, Inoue H, et al. Spatiotemporal pattern of the mouse chondromodulin-I gene expression and its regulatory role in vascular invasion into cartilage during endochondral bone formation[J]. Int J Dev Biol,1999,43(l):39-49.
[17]Kitahara H, Hayami T, Tokunaga K, et al. Chondromodulin-I expression in rat articular cartilage[J]. Arch Histol Cytol,2003,66(3):221-228.
[18]Shukunami C, Yamamoto S, Tanabe T, et al. Generation of multiple transcripts from the chicken chondromodulin-I gene and their expression during embryonic development[J]. FEBS Lett,1999,456(1):165-170.
[19]Dietz U H, Ziegelmeier G, Bittner K, et al. Spatio-temporal distribution of chondromodulin-I mRNA in the chicken embryo:expression during cartilage development and formation of the heart and eye[J]. Dev Dyn,1999,216(3):233-243.
[20]Takao T, Iwaki T, Kondo J, et al. Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes[J], Histochem J,2000,32(9):545-550.
[21]Kusafuka K, Hiraki Y, Shukunami C, et al. Cartilage-specific matrix protein, chondromodulin-I (ChM-I), is a strong angio-inhibitor in endochondral ossification of human neonatal vertebral tissues in vivo:relationship with angiogenic factors in the cartilage[J]. Acta Histochem,2002,104(2):167-175.
[22]Funaki H, Sawaguchi S, Yaoeda K, et al. Expression and localization of angiogenic inhibitory factor, chondromodulin-I, in adult rat eye[J]. Invest Ophthalmol Vis Sci,2001,42(6):1193-1200.
[23]Fukushima A, Funaki H, Yaoeda K, et al. Localization and expression of chondromodulin-I in the rat cornea[J]. Arch Histol Cytol,2003,66(5):445-452.
[24]Nagai T, Sato M, Kutsuna T, et al. Intravenous administration of anti-vascular endothelial growth factor humanized monoclonal antibody bevacizumab improves articular cartilage repair[J]. Arthritis Res Ther,2010,12(5):R178.
[25]Setoguchi K, Misaki Y, Kawahata K, et al. Suppression of T cell responses by chondromodulin I, a cartilage-derived angiogenesis inhibitory factor:therapeutic potential in rheumatoid arthritis[J]. Arthritis Rheum,2004,50(3):828-839.
[26]Hayami T, Shukunami C, Mitsui K, et al. Specific loss of chondromodulin-I gene expression in chondrosarcoma and the suppression of tumor angiogenesis and growth by its recombinant protein in vivo[J]. FEBS Lett,1999,458(3):436-440.
[27]Kusafuka K, Hiraki Y, Shukunami C, et al. Cartilage-specific matrix protein chondromodulin-I is associated with chondroid formation in salivary pleomorphic adenomas:immunohistochemical analysis[J]. Am J Pathol,2001,158(4):1465-1472.
[28]Kauppila L I. Ingrowth of blood vessels in disc degeneration. Angiographic and histological studies of cadaveric spines[J]. J Bone Joint Surg Am,1995,77(1):26-31.
[29]Repanti M, Korovessis P G, Stamatakis M V, et al. Evolution of disc degeneration in lumbar spine:a comparative histological study between herniated and postmortem retrieved disc specimens[J]. J Spinal Disord,1998,11(1):41-45.
[30]Johnson W E, Caterson B, Eisenstein S M, et al. Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro[J]. Spine (Phila Pa 1976),2005,30(10):1139-1147.
[31]Melrose J, Roberts S, Smith S, et al. Increased nerve and blood vessel ingrowth associated with proteoglycan depletion in an ovine anular lesion model of experimental disc degeneration[J]. Spine (Phila Pa 1976),2002,27(12):1278-1285.
[32]Freemont A J, Peacock T E, Goupille P, et al. Nerve ingrowth into diseased intervertebral disc in chronic back pain[J]. Lancet,1997,350(9072):178-181.
[1]Chelberg M K, Banks G M, Geiger D F, et al. Identification of heterogeneous cell populations in normal human intervertebral disc[J]. J Anat,1995,186 (Pt 1):43-53.
[2]Roberts S, Evans H, Trivedi J, et al. Histology and pathology of the human intervertebral disc[J]. J Bone Joint Surg Am,2006,88 Suppl 2:10-14.
[3]Yu J, Fairbank J C, Roberts S, et al. The elastic fiber network of the anulus fibrosus of the normal and scoliotic human intervertebral disc[J]. Spine (Phila Pa 1976),2005,30(16):1815-1820.
[4]Gruber H E, Ingram J A, Hanley E J. Immunolocalization of MMP-19 in the human intervertebral disc:implications for disc aging and degeneration[J]. Biotech Histochem,2005,80(3-4):157-162.
[5]Kozaci L D, Guner A, Oktay G, et al. Alterations in biochemical components of extracellular matrix in intervertebral disc herniation:role of MMP-2 and TIMP-2 in type Ⅱcollagen loss[J]. Cell Biochem Funct,2006,24(5):431-436.
[6]Nerlich A G, Bachmeier B E, Boos N. Expression of fibronectin and TGF-betal mRNA and protein suggest altered regulation of extracellular matrix in degenerated disc tissue[J]. Eur Spine J,2005,14(1):17-26.
[7]Tolonen J, Gronblad M, Vanharanta H, et al. Growth factor expression in degenerated intervertebral disc tissue. An immunohistochemical analysis of transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor[J]. Eur Spine J,2006,15(5):588-596.
[8]Le Maitre C L, Freemont A J, Hoyland J A. The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration[J]. Arthritis Res Ther,2005,7(4):R732-R745.
[9]Ohtori S, Inoue G, Ito T, et al. Tumor necrosis factor-immunoreactive cells and PGP 9.5-immunoreactive nerve fibers in vertebral endplates of patients with discogenic low back Pain and Modic Type 1 or Type 2 changes on MRI[J]. Spine (Phila Pa 1976),2006,31 (9):1026-1031.
[10]Tolonen J, Gronblad M, Vanharanta H, et al. Growth factor expression in degenerated intervertebral disc tissue. An immunohistochemical analysis of transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor[J]. Eur Spine J,2006,15(5):588-596.
[11]Masuda K, Imai Y, Okuma M, et al. Osteogenic protein-1 injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit anular puncture model[J]. Spine (Phila Pa 1976),2006,31(7):742-754.
[12]Le Maitre C L, Freemont A J, Hoyland J A. A preliminary in vitro study into the use of IL-1Ra gene therapy for the inhibition of intervertebral disc degeneration[J]. Int J Exp Pathol,2006,87(1):17-28.
[13]Meisel H J, Ganey T, Hutton W C, et al. Clinical experience in cell-based therapeutics:intervention and outcome[J]. Eur Spine J,2006,15 Suppl 3:S397-S405.
[14]Meisel H J, Siodla V, Ganey T, et al. Clinical experience in cell-based therapeutics:disc chondrocyte transplantation A treatment for degenerated or damaged intervertebral disc[J]. Biomol Eng,2007,24(1):5-21.
[15]Zhao C Q, Wang L M, Jiang L S, et al. The cell biology of intervertebral disc aging and degeneration[J]. Ageing Res Rev,2007,6(3):247-261.
[16]Sakai D, Mochida J, Yamamoto Y, et al. Immortalization of human nucleus pulposus cells by a recombinant SV40 adenovirus vector:establishment of a novel cell line for the study of human nucleus pulposus cells[J]. Spine (Phila Pa 1976),2004,29(14):1515-1523.
[17]Kaneyama S, Nishida K, Takada T, et al. Fas ligand expression on human nucleus pulposus cells decreases with disc degeneration processes[J]. J Orthop Sci,2008,13(2):130-135.
[18]Kluba T, Niemeyer T, Gaissmaier C, et al. Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc:effect of degeneration and culture system on cell phenotype[J]. Spine (Phila Pa 1976),2005,30(24):2743-2748.
[1]Stoscheck C M. Quantitation of protein[J]. Methods Enzymol,1990,182:50-68.
[2]Hiraki Y, Tanaka H, Inoue H, et al. Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes[J]. Biochem Biophys Res Commun,1991,175(3):971-977.
[3]Inoue H, Kondo J, Koike T, et al. Identification of an autocrine chondrocyte colony-stimulating factor:chondromodulin-I stimulates the colony formation of growth plate chondrocytes in agarose culture[J]. Biochem Biophys Res Commun,1997,241 (2):395-400.
[4]Hiraki Y, Kono T, Sato M, et al. Inhibition of DNA synthesis and tube morphogenesis of cultured vascular endothelial cells by chondromodulin-I[J]. FEBS Lett,1997,415(3):321-324.
[5]Funaki H, Sawaguchi S, Yaoeda K, et al. Expression and localization of angiogenic inhibitory factor, chondromodulin-I, in adult rat eye[J]. Invest Ophthalmol Vis Sci,2001,42(6):1193-1200.
[6]Yoshioka M, Yuasa S, Matsumura K, et al. Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis[J]. Nat Med,2006,12(10):1151-1159.
[7]Takao T, Iwaki T, Kondo J, et al. Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes[J]. Histochem J,2000,32(9):545-550.
[8]Mera H, Kawashima H, Yoshizawa T, et al. Chondromodulin-1 directly suppresses growth of human cancer cells[J]. BMC Cancer,2009,9:166.
[9]Hayami T, Funaki H, Yaoeda K, et al. Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis[J]. J Rheumatol,2003,30(10):2207-2217.
[10]Yanagihara I, Yamagata M, Sakai N, et al. Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells[J]. J Bone Miner Res,2000,15(3):421-429.
[11]Hiraki Y, Tanaka H, Inoue H, et al. Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes[J]. Biochem Biophys Res Commun,1991,175(3):971-977.
[12]Azizan A, Holaday N, Neame P J. Post-translational processing of bovine chondromodulin-I[J]. J Biol Chem,2001,276(26):23632-23638.
[13]Nagano T, Yonenobu K, Miyamoto S, et al. Distribution of the basic fibroblast growth factor and its receptor gene expression in normal and degenerated rat intervertebral discs[J]. Spine (Phila Pa 1976),1995,20(18):1972-1978.
[14]Tolonen J, Gronblad M, Vanharanta H, et al. Growth factor expression in degenerated intervertebral disc tissue. An immunohistochemical analysis of transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor[J]. Eur Spine J,2006,15(5):588-596.
[15]Peng B, Hao J, Hou S, et al. Possible pathogenesis of painful intervertebral disc degeneration[J]. Spine (Phila Pa 1976),2006,31(5):560-566.
[16]Pratsinis H, Kletsas D. PDGF, bFGF and IGF-I stimulate the proliferation of intervertebral disc cells in vitro via the activation of the ERK and Akt signaling pathways[J]. Eur Spine J.2007,16(11):1858-1866.
[17]Li X, An H S, Ellman M, et al. Action of fibroblast growth factor-2 on the intervertebral disc[J]. Arthritis Res Ther,2008,10(2):R48.
[18]Ellman M B, An H S, Muddasani P, et al. Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis[J]. Gene,2008,420(1):82-89.
[1]Pfirrmann C W, Metzdorf A, Zanetti M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration[J]. Spine (Phila Pa 1976),2001,26(17):1873-1878.
[2]Hayami T, Funaki H, Yaoeda K, et al. Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis[J]. J Rheumatol,2003,30(10):2207-2217.
[3]Yoshioka M, Yuasa S, Matsumura K, et al. Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis[J]. Nat Med,2006,12(10):1151-1 159.
[4]Hayami T, Shukunami C, Mitsui K, et al. Specific loss of chondromodulin-I gene expression in chondrosarcoma and the suppression of tumor angiogenesis and growth by its recombinant protein in vivo[J]. FEBS Lett,1999,458(3):436-440.
[5]Mera H, Kawashima H, Yoshizawa T, et al. Chondromodulin-1 directly suppresses growth of human cancer cells[J]. BMC Cancer,2009,9:166.
[6]Takao T, Iwaki T, Kondo J, et al. Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes[J]. Histochem J,2000,32(9):545-550.
[7]Weiler C, Nerlich A G, Bachmeier B E, et al. Expression and distribution of tumor necrosis factor alpha in human lumbar intervertebral discs:a study in surgical specimen and autopsy controls[J]. Spine (Phila Pa 1976),2005,30(1):44-53,54.
[8]Takao T, Iwaki T, Kondo J, et al. Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes[J]. Histochem J,2000,32(9):545-550.
[2]Funaki H, Sawaguchi S, Yaoeda K, et al. Expression and localization of angiogenic inhibitory factor, chondromodulin-I, in adult rat eye[J]. Invest Ophthalmol Vis Sci,2001,42(6):1193-1200.
[3]Yoshioka M, Yuasa S, Matsumura K, et al. Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis[J]. Nat Med,2006,12(10):1151-1159.
[4]Hayami T, Funaki H, Yaoeda K, et al. Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis[J]. J Rheumatol,2003,30(10):2207-2217.
[5]Mera H, Kawashima H, Yoshizawa T, et al. Chondromodulin-1 directly suppresses growth of human cancer cells[J]. BMC Cancer,2009,9:166.
[6]Freemont A J, Peacock T E, Goupille P, et al. Nerve ingrowth into diseased intervertebral disc in chronic back pain[J]. Lancet,1997,350(9072):178-181.
[7]Peng B, Hao J, Hou S, et al. Possible pathogenesis of painful intervertebral disc degeneration[J]. Spine (Phila Pa 1976),2006,31(5):560-566.
[8]Hiraki Y, Tanaka H, Inoue H, et al. Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes[J]. Biochem Biophys Res Commun,1991,175(3):971-977.
[9]Inoue H, Kondo J, Koike T, et al. Identification of an autocrine chondrocyte colony-stimulating factor:chondromodulin-I stimulates the colony formation of growth plate chondrocytes in agarose culture[J]. Biochem Biophys Res Commun,1997,241(2):395-400.
[10]Hiraki Y, Kono T, Sato M, et al. Inhibition of DNA synthesis and tube morphogenesis of cultured vascular endothelial cells by chondromodulin-I[J]. FEBS Lett,1997,415(3):321-324.
[11]Yanagihara I, Yamagata M, Sakai N, et al. Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells[J]. J Bone Miner Res,2000,15(3):421-429.
[12]Hiraki Y, Tanaka H, Inoue H, et al. Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes[J]. Biochem Biophys Res Commun,1991,175(3):971-977.
[13]Azizan A, Holaday N, Neame P J. Post-translational processing of bovine chondromodulin-I[J]. J Biol Chem,2001,276(26):23632-23638.
[14]Aoyama T, Okamoto T, Nagayama S, et al. Methylation in the core-promoter region of the chondromodulin-I gene determines the cell-specific expression by regulating the binding of transcriptional activator Sp3[J]. J Biol Chem,2004,279(27):28789-28797.
[15]Aoyama T, Okamoto T, Kohno Y, et al. Cell-specific epigenetic regulation of ChM-I gene expression:crosstalk between DNA methylation and histone acetylation[J]. Biochem Biophys Res Commun,2008,365(1):124-130.
[16]Shukunami C, Iyama K, Inoue H, et al. Spatiotemporal pattern of the mouse chondromodulin-I gene expression and its regulatory role in vascular invasion into cartilage during endochondral bone formation[J]. Int J Dev Biol,1999,43(l):39-49.
[17]Kitahara H, Hayami T, Tokunaga K, et al. Chondromodulin-I expression in rat articular cartilage[J]. Arch Histol Cytol,2003,66(3):221-228.
[18]Shukunami C, Yamamoto S, Tanabe T, et al. Generation of multiple transcripts from the chicken chondromodulin-I gene and their expression during embryonic development[J]. FEBS Lett,1999,456(1):165-170.
[19]Dietz U H, Ziegelmeier G, Bittner K, et al. Spatio-temporal distribution of chondromodulin-I mRNA in the chicken embryo:expression during cartilage development and formation of the heart and eye[J]. Dev Dyn,1999,216(3):233-243.
[20]Takao T, Iwaki T, Kondo J, et al. Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes[J], Histochem J,2000,32(9):545-550.
[21]Kusafuka K, Hiraki Y, Shukunami C, et al. Cartilage-specific matrix protein, chondromodulin-I (ChM-I), is a strong angio-inhibitor in endochondral ossification of human neonatal vertebral tissues in vivo:relationship with angiogenic factors in the cartilage[J]. Acta Histochem,2002,104(2):167-175.
[22]Funaki H, Sawaguchi S, Yaoeda K, et al. Expression and localization of angiogenic inhibitory factor, chondromodulin-I, in adult rat eye[J]. Invest Ophthalmol Vis Sci,2001,42(6):1193-1200.
[23]Fukushima A, Funaki H, Yaoeda K, et al. Localization and expression of chondromodulin-I in the rat cornea[J]. Arch Histol Cytol,2003,66(5):445-452.
[24]Nagai T, Sato M, Kutsuna T, et al. Intravenous administration of anti-vascular endothelial growth factor humanized monoclonal antibody bevacizumab improves articular cartilage repair[J]. Arthritis Res Ther,2010,12(5):R178.
[25]Setoguchi K, Misaki Y, Kawahata K, et al. Suppression of T cell responses by chondromodulin I, a cartilage-derived angiogenesis inhibitory factor:therapeutic potential in rheumatoid arthritis[J]. Arthritis Rheum,2004,50(3):828-839.
[26]Hayami T, Shukunami C, Mitsui K, et al. Specific loss of chondromodulin-I gene expression in chondrosarcoma and the suppression of tumor angiogenesis and growth by its recombinant protein in vivo[J]. FEBS Lett,1999,458(3):436-440.
[27]Kusafuka K, Hiraki Y, Shukunami C, et al. Cartilage-specific matrix protein chondromodulin-I is associated with chondroid formation in salivary pleomorphic adenomas:immunohistochemical analysis[J]. Am J Pathol,2001,158(4):1465-1472.
[28]Kauppila L I. Ingrowth of blood vessels in disc degeneration. Angiographic and histological studies of cadaveric spines[J]. J Bone Joint Surg Am,1995,77(1):26-31.
[29]Repanti M, Korovessis P G, Stamatakis M V, et al. Evolution of disc degeneration in lumbar spine:a comparative histological study between herniated and postmortem retrieved disc specimens[J]. J Spinal Disord,1998,11(1):41-45.
[30]Johnson W E, Caterson B, Eisenstein S M, et al. Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro[J]. Spine (Phila Pa 1976),2005,30(10):1139-1147.
[31]Melrose J, Roberts S, Smith S, et al. Increased nerve and blood vessel ingrowth associated with proteoglycan depletion in an ovine anular lesion model of experimental disc degeneration[J]. Spine (Phila Pa 1976),2002,27(12):1278-1285.
[32]Freemont A J, Peacock T E, Goupille P, et al. Nerve ingrowth into diseased intervertebral disc in chronic back pain[J]. Lancet,1997,350(9072):178-181.
[1]Chelberg M K, Banks G M, Geiger D F, et al. Identification of heterogeneous cell populations in normal human intervertebral disc[J]. J Anat,1995,186 (Pt 1):43-53.
[2]Roberts S, Evans H, Trivedi J, et al. Histology and pathology of the human intervertebral disc[J]. J Bone Joint Surg Am,2006,88 Suppl 2:10-14.
[3]Yu J, Fairbank J C, Roberts S, et al. The elastic fiber network of the anulus fibrosus of the normal and scoliotic human intervertebral disc[J]. Spine (Phila Pa 1976),2005,30(16):1815-1820.
[4]Gruber H E, Ingram J A, Hanley E J. Immunolocalization of MMP-19 in the human intervertebral disc:implications for disc aging and degeneration[J]. Biotech Histochem,2005,80(3-4):157-162.
[5]Kozaci L D, Guner A, Oktay G, et al. Alterations in biochemical components of extracellular matrix in intervertebral disc herniation:role of MMP-2 and TIMP-2 in type Ⅱcollagen loss[J]. Cell Biochem Funct,2006,24(5):431-436.
[6]Nerlich A G, Bachmeier B E, Boos N. Expression of fibronectin and TGF-betal mRNA and protein suggest altered regulation of extracellular matrix in degenerated disc tissue[J]. Eur Spine J,2005,14(1):17-26.
[7]Tolonen J, Gronblad M, Vanharanta H, et al. Growth factor expression in degenerated intervertebral disc tissue. An immunohistochemical analysis of transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor[J]. Eur Spine J,2006,15(5):588-596.
[8]Le Maitre C L, Freemont A J, Hoyland J A. The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration[J]. Arthritis Res Ther,2005,7(4):R732-R745.
[9]Ohtori S, Inoue G, Ito T, et al. Tumor necrosis factor-immunoreactive cells and PGP 9.5-immunoreactive nerve fibers in vertebral endplates of patients with discogenic low back Pain and Modic Type 1 or Type 2 changes on MRI[J]. Spine (Phila Pa 1976),2006,31 (9):1026-1031.
[10]Tolonen J, Gronblad M, Vanharanta H, et al. Growth factor expression in degenerated intervertebral disc tissue. An immunohistochemical analysis of transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor[J]. Eur Spine J,2006,15(5):588-596.
[11]Masuda K, Imai Y, Okuma M, et al. Osteogenic protein-1 injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit anular puncture model[J]. Spine (Phila Pa 1976),2006,31(7):742-754.
[12]Le Maitre C L, Freemont A J, Hoyland J A. A preliminary in vitro study into the use of IL-1Ra gene therapy for the inhibition of intervertebral disc degeneration[J]. Int J Exp Pathol,2006,87(1):17-28.
[13]Meisel H J, Ganey T, Hutton W C, et al. Clinical experience in cell-based therapeutics:intervention and outcome[J]. Eur Spine J,2006,15 Suppl 3:S397-S405.
[14]Meisel H J, Siodla V, Ganey T, et al. Clinical experience in cell-based therapeutics:disc chondrocyte transplantation A treatment for degenerated or damaged intervertebral disc[J]. Biomol Eng,2007,24(1):5-21.
[15]Zhao C Q, Wang L M, Jiang L S, et al. The cell biology of intervertebral disc aging and degeneration[J]. Ageing Res Rev,2007,6(3):247-261.
[16]Sakai D, Mochida J, Yamamoto Y, et al. Immortalization of human nucleus pulposus cells by a recombinant SV40 adenovirus vector:establishment of a novel cell line for the study of human nucleus pulposus cells[J]. Spine (Phila Pa 1976),2004,29(14):1515-1523.
[17]Kaneyama S, Nishida K, Takada T, et al. Fas ligand expression on human nucleus pulposus cells decreases with disc degeneration processes[J]. J Orthop Sci,2008,13(2):130-135.
[18]Kluba T, Niemeyer T, Gaissmaier C, et al. Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc:effect of degeneration and culture system on cell phenotype[J]. Spine (Phila Pa 1976),2005,30(24):2743-2748.
[1]Stoscheck C M. Quantitation of protein[J]. Methods Enzymol,1990,182:50-68.
[2]Hiraki Y, Tanaka H, Inoue H, et al. Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes[J]. Biochem Biophys Res Commun,1991,175(3):971-977.
[3]Inoue H, Kondo J, Koike T, et al. Identification of an autocrine chondrocyte colony-stimulating factor:chondromodulin-I stimulates the colony formation of growth plate chondrocytes in agarose culture[J]. Biochem Biophys Res Commun,1997,241 (2):395-400.
[4]Hiraki Y, Kono T, Sato M, et al. Inhibition of DNA synthesis and tube morphogenesis of cultured vascular endothelial cells by chondromodulin-I[J]. FEBS Lett,1997,415(3):321-324.
[5]Funaki H, Sawaguchi S, Yaoeda K, et al. Expression and localization of angiogenic inhibitory factor, chondromodulin-I, in adult rat eye[J]. Invest Ophthalmol Vis Sci,2001,42(6):1193-1200.
[6]Yoshioka M, Yuasa S, Matsumura K, et al. Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis[J]. Nat Med,2006,12(10):1151-1159.
[7]Takao T, Iwaki T, Kondo J, et al. Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes[J]. Histochem J,2000,32(9):545-550.
[8]Mera H, Kawashima H, Yoshizawa T, et al. Chondromodulin-1 directly suppresses growth of human cancer cells[J]. BMC Cancer,2009,9:166.
[9]Hayami T, Funaki H, Yaoeda K, et al. Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis[J]. J Rheumatol,2003,30(10):2207-2217.
[10]Yanagihara I, Yamagata M, Sakai N, et al. Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells[J]. J Bone Miner Res,2000,15(3):421-429.
[11]Hiraki Y, Tanaka H, Inoue H, et al. Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes[J]. Biochem Biophys Res Commun,1991,175(3):971-977.
[12]Azizan A, Holaday N, Neame P J. Post-translational processing of bovine chondromodulin-I[J]. J Biol Chem,2001,276(26):23632-23638.
[13]Nagano T, Yonenobu K, Miyamoto S, et al. Distribution of the basic fibroblast growth factor and its receptor gene expression in normal and degenerated rat intervertebral discs[J]. Spine (Phila Pa 1976),1995,20(18):1972-1978.
[14]Tolonen J, Gronblad M, Vanharanta H, et al. Growth factor expression in degenerated intervertebral disc tissue. An immunohistochemical analysis of transforming growth factor beta, fibroblast growth factor and platelet-derived growth factor[J]. Eur Spine J,2006,15(5):588-596.
[15]Peng B, Hao J, Hou S, et al. Possible pathogenesis of painful intervertebral disc degeneration[J]. Spine (Phila Pa 1976),2006,31(5):560-566.
[16]Pratsinis H, Kletsas D. PDGF, bFGF and IGF-I stimulate the proliferation of intervertebral disc cells in vitro via the activation of the ERK and Akt signaling pathways[J]. Eur Spine J.2007,16(11):1858-1866.
[17]Li X, An H S, Ellman M, et al. Action of fibroblast growth factor-2 on the intervertebral disc[J]. Arthritis Res Ther,2008,10(2):R48.
[18]Ellman M B, An H S, Muddasani P, et al. Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis[J]. Gene,2008,420(1):82-89.
[1]Pfirrmann C W, Metzdorf A, Zanetti M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration[J]. Spine (Phila Pa 1976),2001,26(17):1873-1878.
[2]Hayami T, Funaki H, Yaoeda K, et al. Expression of the cartilage derived anti-angiogenic factor chondromodulin-I decreases in the early stage of experimental osteoarthritis[J]. J Rheumatol,2003,30(10):2207-2217.
[3]Yoshioka M, Yuasa S, Matsumura K, et al. Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis[J]. Nat Med,2006,12(10):1151-1 159.
[4]Hayami T, Shukunami C, Mitsui K, et al. Specific loss of chondromodulin-I gene expression in chondrosarcoma and the suppression of tumor angiogenesis and growth by its recombinant protein in vivo[J]. FEBS Lett,1999,458(3):436-440.
[5]Mera H, Kawashima H, Yoshizawa T, et al. Chondromodulin-1 directly suppresses growth of human cancer cells[J]. BMC Cancer,2009,9:166.
[6]Takao T, Iwaki T, Kondo J, et al. Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes[J]. Histochem J,2000,32(9):545-550.
[7]Weiler C, Nerlich A G, Bachmeier B E, et al. Expression and distribution of tumor necrosis factor alpha in human lumbar intervertebral discs:a study in surgical specimen and autopsy controls[J]. Spine (Phila Pa 1976),2005,30(1):44-53,54.
[8]Takao T, Iwaki T, Kondo J, et al. Immunohistochemistry of chondromodulin-I in the human intervertebral discs with special reference to the degenerative changes[J]. Histochem J,2000,32(9):545-550.