自體富血小板血漿干預兔早期椎間盤退變的初步研究_《中國修復重建外科雜志》

作者：

胡新鋒 , 王宸 , 芮云峰

東南大學附屬中大醫院骨科（南京，210009）;

關鍵詞：

自體富血小板血漿早期椎間盤退變生物治療 MRI Ⅱ型膠原兔

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目的富血小板血漿（platelet-rich plasma，PRP）具有刺激椎間盤細胞增殖、促進細胞外基質合成代謝及抑制纖維環細胞凋亡等作用。通過觀察自體PRP干預兔早期椎間盤退變，明確其治療效果，為臨床應用提供理論依據。方法取健康成年新西蘭大白兔45只，體重2.5～3.0 kg，雌雄不限；隨機分為實驗組、對照組、假手術組（n=15）。取實驗組兔耳中央動脈血，采用Landesberg等方法制備PRP，同時對全血及PRP行血小板計數。實驗組及對照組采用纖維環針刺法建立L4、5及L5、6椎間盤退變模型，造模2周后于L4、5及L5、6椎間隙分別注入100 ?L自體PRP及100 ?L PBS液；假手術組僅分離暴露椎間盤，不作處理。觀察實驗動物造模后一般情況；造模2周及干預1、2周時各組取5只實驗動物行腰椎MRI、HE染色及Ⅱ型膠原免疫組織化學染色觀察，腰椎MRI退變程度分級及Ⅱ型膠原陽性積分吸光度（IA）值檢測。結果兔PRP中血小板計數約為外周血的4.92倍。實驗動物均存活至實驗完成。造模2周時，與假手術組相比，實驗組和對照組椎間盤信號降低，髓核細胞減少，基質退變，Ⅱ型膠原表達降低。腰椎MRI退變程度分級及Ⅱ型膠原陽性IA值結果顯示，各時間點實驗組、對照組與假手術組相比差異均有統計學意義（P lt; 0.05），干預1、2周時，實驗組MRI退變程度分級顯著低于對照組（P lt; 0.05），但仍與假手術組有差異（P lt; 0.05）；干預1、2周時，實驗組髓核細胞及軟骨樣基質較對照組增多，基質纖維化程度輕，Ⅱ型膠原表達明顯強于對照組（P lt; 0.05）。結論椎間盤內注射自體PRP可終止甚至一定程度逆轉兔早期椎間盤退變，可能與PRP含有多種生長因子調控細胞功能、改善組織微環境、促進組織再生修復有關。

引用本文： 胡新鋒,王宸,芮云峰. 自體富血小板血漿干預兔早期椎間盤退變的初步研究. 中國修復重建外科雜志, 2012, 26(8): 977-983. doi: 復制

1.	Adams MA, Roughley PJ. What is intervertebral disc degeneration, and what causes it. Spine (Phila Pa 1976), 2006, 31(18): 2151-2161.
2.	Karppinen J, Shen FH, Luk KD, et al. Management of degenerative disk disease and chronic low back pain. Orthop Clin North Am, 2011, 42(4): 513-528.
3.	Masuda K. Biological repair of the degenerated intervertebral disc by the injection of growth factors. Eur Spine, 2008, 17 Suppl 4: 441-451.
4.	耿震, 王宸, 周海洋. 富血小板血漿對肌腱愈合影響的實驗研究. 中國修復重建外科雜志, 2011, 25(3): 344-348.
5.	Chen S, Emery SE, Pei M. Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-beta1: a potential application of tissue specific stem cells in disc regeneration. Spine (Phila Pa 1976), 2009, 34(12): 1272-1280.
6.	Gruber HE, Norton HJ, Hanley EN Jr. Anti-apoptotic effects of IGF-1 and PDGF on human interverterbral disc cells in vitro. Spine (Phila Pa 1976), 2000, 25(17): 2153-2157.
7.	Osada R, Ohshima H, Ishihara H, et al. Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs. J Orthop Res, 1996, 14(5): 690-699.
8.	Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation. J Oral Maxillofac Surg, 2000, 58(3): 297-300.
9.	Chen WH, Lo WC, Lee JJ, et al. Tissue-engineered intervertebral disc and chondrogenesis using human nucleus pulposus regulated through TGF-beta1 in platelet-rich plasma. J Cell Physiol, 2006, 209(3): 744-754.
10.	Masuda K, Aota Y, Muehleman C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976), 2005, 30(1): 5-41.
11.	Eppley BL, Woodell JE, Higgin J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plast Reconstr Surg, 2004, 114(6): 1502-1508.
12.	張聞力, 李濤, 龔全, 等. 三種方法建立兔椎間盤退變模型的比較研究. 中國修復重建外科雜志, 2010, 24(1): 41-45.
13.	張聞力, 劉浩, 李壇珠. 椎間盤退變動物模型的研究進展. 中國修復重建外科雜志, 2007, 21(11): 1259-1262.
14.	Kim KS, Yoon ST, Li J, et al. Disc degeneration in the rabbit: a biochemical and radiological comparison between four disc injury models. Spine (Phila Pa 1976), 2005, 30(1): 33-37.
15.	桂柯科, 尹望平, 張飚, 等. 纖維環穿刺法建立兔椎間盤退變模型. 中國矯形外科雜志, 2010, 18(21): 1814-1816.
16.	Urban JP, Roberts S. Degeneration of the intervertebral disc. Arthritis Res Ther, 2003, 5(3): 120-130.
17.	Thompson JP, Pearce RH, Schechter MT, et al. Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine (Phila Pa 1976), 1990, 15(5): 411-415.
18.	Pfirrmann CW, Metzdorf A, Zanetti M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine (Phila Pa 1976), 2001, 26(17): 1873-1878.
19.	Zhang Y, Chee A, Thonar EJ, et al. Intervertebral disk repair by protein, gene, or cell injection: a framework for rehabilitation-focused biologics in the spine. PMR, 2011, 3(6 Suppl 1): S88-94.
20.	Nagae M, Ikeda T, Mikami Y, et al. Intervertebral disc regeneration using platelet-rich plasma and biodegradable gelatin hydrogel microspheres. Tissue Eng, 2007, 13(1): 147-158.
21.	Sawamura K, Ikeda T, Nagae M, et al. Characterization of in vivo effects of platelet-rich plasma and biodegradable gelatin hydrogel microspheres on degenerated intervertebral discs. Tissue Eng Part A, 2009, 15(12): 3719-3727.
22.	Chen WH, Liu HY, Lo WC, et al. Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma. Biomaterials, 2009, 30(29): 5523-5533.
23.	Akeda K, An HS, Pichika R, et al. Platelet-rich plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads. Spine (Phila Pa 1976), 2006, 31(9): 959-966.
24.	Gruber HE, Hanley EN Jr. Analysis of aging and degeneration of the human intervertebral disc. Comparison of surgical specimens with normal controls. Spine (Phila Pa 1976), 1998, 23(7): 751-757.

1. Adams MA, Roughley PJ. What is intervertebral disc degeneration, and what causes it. Spine (Phila Pa 1976), 2006, 31(18): 2151-2161.
2. Karppinen J, Shen FH, Luk KD, et al. Management of degenerative disk disease and chronic low back pain. Orthop Clin North Am, 2011, 42(4): 513-528.
3. Masuda K. Biological repair of the degenerated intervertebral disc by the injection of growth factors. Eur Spine, 2008, 17 Suppl 4: 441-451.
4. 耿震, 王宸, 周海洋. 富血小板血漿對肌腱愈合影響的實驗研究. 中國修復重建外科雜志, 2011, 25(3): 344-348.
5. Chen S, Emery SE, Pei M. Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-beta1: a potential application of tissue specific stem cells in disc regeneration. Spine (Phila Pa 1976), 2009, 34(12): 1272-1280.
6. Gruber HE, Norton HJ, Hanley EN Jr. Anti-apoptotic effects of IGF-1 and PDGF on human interverterbral disc cells in vitro. Spine (Phila Pa 1976), 2000, 25(17): 2153-2157.
7. Osada R, Ohshima H, Ishihara H, et al. Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs. J Orthop Res, 1996, 14(5): 690-699.
8. Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation. J Oral Maxillofac Surg, 2000, 58(3): 297-300.
9. Chen WH, Lo WC, Lee JJ, et al. Tissue-engineered intervertebral disc and chondrogenesis using human nucleus pulposus regulated through TGF-beta1 in platelet-rich plasma. J Cell Physiol, 2006, 209(3): 744-754.
10. Masuda K, Aota Y, Muehleman C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976), 2005, 30(1): 5-41.
11. Eppley BL, Woodell JE, Higgin J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plast Reconstr Surg, 2004, 114(6): 1502-1508.
12. 張聞力, 李濤, 龔全, 等. 三種方法建立兔椎間盤退變模型的比較研究. 中國修復重建外科雜志, 2010, 24(1): 41-45.
13. 張聞力, 劉浩, 李壇珠. 椎間盤退變動物模型的研究進展. 中國修復重建外科雜志, 2007, 21(11): 1259-1262.
14. Kim KS, Yoon ST, Li J, et al. Disc degeneration in the rabbit: a biochemical and radiological comparison between four disc injury models. Spine (Phila Pa 1976), 2005, 30(1): 33-37.
15. 桂柯科, 尹望平, 張飚, 等. 纖維環穿刺法建立兔椎間盤退變模型. 中國矯形外科雜志, 2010, 18(21): 1814-1816.
16. Urban JP, Roberts S. Degeneration of the intervertebral disc. Arthritis Res Ther, 2003, 5(3): 120-130.
17. Thompson JP, Pearce RH, Schechter MT, et al. Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine (Phila Pa 1976), 1990, 15(5): 411-415.
18. Pfirrmann CW, Metzdorf A, Zanetti M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine (Phila Pa 1976), 2001, 26(17): 1873-1878.
19. Zhang Y, Chee A, Thonar EJ, et al. Intervertebral disk repair by protein, gene, or cell injection: a framework for rehabilitation-focused biologics in the spine. PMR, 2011, 3(6 Suppl 1): S88-94.
20. Nagae M, Ikeda T, Mikami Y, et al. Intervertebral disc regeneration using platelet-rich plasma and biodegradable gelatin hydrogel microspheres. Tissue Eng, 2007, 13(1): 147-158.
21. Sawamura K, Ikeda T, Nagae M, et al. Characterization of in vivo effects of platelet-rich plasma and biodegradable gelatin hydrogel microspheres on degenerated intervertebral discs. Tissue Eng Part A, 2009, 15(12): 3719-3727.
22. Chen WH, Liu HY, Lo WC, et al. Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma. Biomaterials, 2009, 30(29): 5523-5533.
23. Akeda K, An HS, Pichika R, et al. Platelet-rich plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads. Spine (Phila Pa 1976), 2006, 31(9): 959-966.
24. Gruber HE, Hanley EN Jr. Analysis of aging and degeneration of the human intervertebral disc. Comparison of surgical specimens with normal controls. Spine (Phila Pa 1976), 1998, 23(7): 751-757.

《中國修復重建外科雜志》

自體富血小板血漿干預兔早期椎間盤退變的初步研究

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《中國修復重建外科雜志》

自體富血小板血漿干預兔早期椎間盤退變的初步研究

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

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Content

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