EXPRESSION OF p16<sup>INK4a</sup> IN NUCLEUS PULPOSUS AND ITS EFFECT ON DEGENERATED INTERVERTEBRAL DISCS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

 WANGYuntao , WUXiaotao , WANGFeng , ZHUHouyi , WANGXiaohu , SHIRui

Department of Orthopedics, Zhongda Hospital, Southeast University, Nanjing Jiangsu, 210009, P. R. China;

Corresponding author：

WANGYuntao, Email: wangyttod@seu.edu.cn

Keywords：

Premature senescence; p16^INK4a; Nucleus pulposus cells; Intervertebral disc degeneration

DOI：

10.7507/1002-1892.20140328

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Objective To investigate the expression of p16^INK4a in nucleus pulposus (NP) and to clarify its relationship with intervertebral disc degeneration so as to provide evidence for biological repair of intervertebral disc. Methods The NP specimens were obtained from 17 patients with intervertebral disc degeneration undergoing discectomy, who aged 40-50 years (mean, 45.4 years). Based on the preoperative MRI, there were 10 cases of grade Ⅲ degeneration, and 7 cases of grade IV degeneration. Cell senescence was evaluated by detecting senescence-associated β-galactosidase (SA-β-gal) activity. Senescence marker (p16^INK4a) and disc degeneration markers [A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS 5), Aggrecan, and Sryrelated HMG box transcri ption factor 9 (Sox-9)] were determined in the NP specimens with immunohistochemistry and Western blot. The correlation between ADAMTS 5 and p16^INK4a was analyzed. Results Clustered distribution of green SA-β-gal-positive cells was seen in the NP with grade Ⅲ and IV degeneration. A few single round SA-β-gal-positive NP cells (NPCs) wrapped by the layered extracellular matrix were also seen in the NP with grade Ⅲ degeneration. It was difficult to see single distribution of NPCs in the NP with grade IV degeneration. The percentage of SA-β-gal-positive cells was 22.7%±5.4% and 37.1%±7.6% in the NP with grade Ⅲ and IV degeneration respectively, showing significant difference (t=-9.666, P=0.000). The percentages of p16^INK4a-positive and ADAMTS 5-positive NPCs in the NP with grade IV degeneration were significantly higher than those with grade Ⅲ degeneration (P<0.05). The percentages of Aggrecan-positive and Sox-9-positive NPCs in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The protein expressions of Aggrecan and Sox-9 in the NP with grade IV degeneration were significantly lower than those in the NP with grade Ⅲ degeneration (P<0.05). The NP with grade IV degeneration showed significantly higher protein expressions of p16^INK4a and ADAMTS 5 (P<0.05). Importantly, there was a good correlation between p16^INK4a and ADAMTS 5 protein expressions (r=0.908, P=0.000). Conclusion Premature senescent NPCs increase in the NP with the advancing disc degeneration. The expression of p16^INK4a and its association with degeneration grades suggest that the p16^INK4a may play a significant role in the pathogenesis of intervertebral disc degeneration.

Citation： WANGYuntao, WUXiaotao, WANGFeng, ZHUHouyi, WANGXiaohu, SHIRui. EXPRESSION OF p16^INK4a IN NUCLEUS PULPOSUS AND ITS EFFECT ON DEGENERATED INTERVERTEBRAL DISCS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(12): 1514-1518. doi: 10.7507/1002-1892.20140328 Copy

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2.	王锋, 吴小涛, 王运涛, 等. 干细胞移植修复椎间盘退变的研究现状. 中国修复重建外科杂志, 2013, 27(5):568-572.
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5.	王锋, 吴小涛, 王运涛, 等. 非接触共培养条件下人BMSCs对氧化应激环境中退变髓核细胞凋亡的保护研究. 中国修复重建外科杂志, 2010, 24(4):391-398.
6.	Kim KW, Chung HN, Ha KY, et al. Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs. Spine J, 2009, 9(8):658-666.
7.	Le Maitre CL, Freemont AJ, Hoyland JA. Accelerated cellular senescence in degenerate intervertebral discs:a possible role in the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther, 2007, 9(3):R45.
8.	Park JS, Park JB, Park IJ, et al. Accelerated premature stress-induced senescence of young annulus fibrosus cells of rats by high glucoseinduced oxidative stress. Int Orthop, 2014, 38(6):1311-1320.
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10.	王璞, 陈露萍, 李清泉, 等. 细胞衰老与衰老细胞. 生物化学与生物物理进展, 2012, 39(3):257-263.
11.	Gruber HE, Norton HJ, Ingram JA, et al. The SOX9 transcri ption factor in the human disc:decreased immunolocalization with age and disc degeneration. Spine, 2005, 30(6):625-630.
12.	王珏, 黄宗强, 王义生, 等. Sox9基因对兔腰椎间盘退变的作用. 中华实验外科杂志, 2010, 27(7):967-969.
13.	Wang YT, Wu XT, Chen H, et al. Endoscopy-assisted posterior lumbar interbody fusion in a single segment. J Clin Neurosci, 2014, 21(2):287-292.
14.	Ciapetti G, Granchi D, Devescovi V, et al. Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs. Eur Spine J, 2012, 21(Suppl 1):S10-19.
15.	Wang YT, Wu XT, Wang F. Regeneration potential and mechanism of bone marrow mesenchymal stem cell transplation for treating intervertebral disc degeneration. J Orthop Sci, 2010, 15(6):707-719.
16.	Burova E, Borodkina A, Shatrova A, et al. Sublethal oxidative stress induces the premature senescence of human mesenchymal stem cells derived from endometrium. Oxid Med Cell Longev, 2013, 2013:474931.
17.	张涛, 钱济先, 姬振伟, 等. 沉默p53和p21基因延缓髓核细胞衰老退变实验研究. 中国修复重建外科杂志, 2012, 26(7):796-802.
18.	Pfirrmann CW, Metzdorf A, Zanetti M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine, 2001, 26(17):1873-1878.
19.	Zhao CQ, Wang LM, Jiang LS, et al. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev, 2007, 6(3):247-261.
20.	Bedore J, Sha W, McCann MR, et al. Impaired intervertebral disc development and premature disc degeneration in mice with notochordspecific deletion of CCN2. Arthritis Rheum, 2013, 65(10):2634-2644.
21.	Hao H, Chen G, Liu J, et al. Culturing on Wharton's jelly extract delays mesenchymal stem cell senescence through p53 and p16^INK4a/pRb pathways. PLoS One, 2013, 8(3):e58314.

1. Tang X, Jing L, Chen J. Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging. PLoS One, 2012, 7(12): e52020.
2. 王锋, 吴小涛, 王运涛, 等. 干细胞移植修复椎间盘退变的研究现状. 中国修复重建外科杂志, 2013, 27(5):568-572.
3. Alkhatib B, Rosenzweig DH, Krock E, et al. Acute mechanical injury of the human intervertebral disc:link to degeneration and pain. Eur Cell Mater, 2014, 28:98-111.
4. Risbud MV, Shapiro IM. Role of cytokines in intervertebral disc degeneration: pain and disc content. Nat Rev Rheumatol, 2014, 10(1):44-56.
5. 王锋, 吴小涛, 王运涛, 等. 非接触共培养条件下人BMSCs对氧化应激环境中退变髓核细胞凋亡的保护研究. 中国修复重建外科杂志, 2010, 24(4):391-398.
6. Kim KW, Chung HN, Ha KY, et al. Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs. Spine J, 2009, 9(8):658-666.
7. Le Maitre CL, Freemont AJ, Hoyland JA. Accelerated cellular senescence in degenerate intervertebral discs:a possible role in the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther, 2007, 9(3):R45.
8. Park JS, Park JB, Park IJ, et al. Accelerated premature stress-induced senescence of young annulus fibrosus cells of rats by high glucoseinduced oxidative stress. Int Orthop, 2014, 38(6):1311-1320.
9. Kim KW, Ha KY, Lee JS, et al. Senescence of nucleus pulposus chondrocytes in human intervertebral discs. Asian Spine J, 2008, 2(1):1-8.
10. 王璞, 陈露萍, 李清泉, 等. 细胞衰老与衰老细胞. 生物化学与生物物理进展, 2012, 39(3):257-263.
11. Gruber HE, Norton HJ, Ingram JA, et al. The SOX9 transcri ption factor in the human disc:decreased immunolocalization with age and disc degeneration. Spine, 2005, 30(6):625-630.
12. 王珏, 黄宗强, 王义生, 等. Sox9基因对兔腰椎间盘退变的作用. 中华实验外科杂志, 2010, 27(7):967-969.
13. Wang YT, Wu XT, Chen H, et al. Endoscopy-assisted posterior lumbar interbody fusion in a single segment. J Clin Neurosci, 2014, 21(2):287-292.
14. Ciapetti G, Granchi D, Devescovi V, et al. Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs. Eur Spine J, 2012, 21(Suppl 1):S10-19.
15. Wang YT, Wu XT, Wang F. Regeneration potential and mechanism of bone marrow mesenchymal stem cell transplation for treating intervertebral disc degeneration. J Orthop Sci, 2010, 15(6):707-719.
16. Burova E, Borodkina A, Shatrova A, et al. Sublethal oxidative stress induces the premature senescence of human mesenchymal stem cells derived from endometrium. Oxid Med Cell Longev, 2013, 2013:474931.
17. 张涛, 钱济先, 姬振伟, 等. 沉默p53和p21基因延缓髓核细胞衰老退变实验研究. 中国修复重建外科杂志, 2012, 26(7):796-802.
18. Pfirrmann CW, Metzdorf A, Zanetti M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine, 2001, 26(17):1873-1878.
19. Zhao CQ, Wang LM, Jiang LS, et al. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev, 2007, 6(3):247-261.
20. Bedore J, Sha W, McCann MR, et al. Impaired intervertebral disc development and premature disc degeneration in mice with notochordspecific deletion of CCN2. Arthritis Rheum, 2013, 65(10):2634-2644.
21. Hao H, Chen G, Liu J, et al. Culturing on Wharton's jelly extract delays mesenchymal stem cell senescence through p53 and p16^INK4a/pRb pathways. PLoS One, 2013, 8(3):e58314.

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EXPRESSION OF p16^INK4a IN NUCLEUS PULPOSUS AND ITS EFFECT ON DEGENERATED INTERVERTEBRAL DISCS

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EXPRESSION OF p16INK4a IN NUCLEUS PULPOSUS AND ITS EFFECT ON DEGENERATED INTERVERTEBRAL DISCS

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EXPRESSION OF p16^INK4a IN NUCLEUS PULPOSUS AND ITS EFFECT ON DEGENERATED INTERVERTEBRAL DISCS