中国修复重建外科杂志

中国修复重建外科杂志

不同代次髓核细胞生物学特性的比较研究

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目的通过兔髓核细胞(nucleus pulposus cells,NPCs)自然传代培养退变模型,观察不同代次 NPCs 生物学特性。方法取 6~8 周龄新西兰大白兔(体质量 1.5~2.5 kg)胸腰椎节段髓核组织,采用酶消化法获取原代 NPCs,计为 P0 代;胰蛋白酶消化法传代获取第 1~3 代 NPCs,依次计为 P1、P2、P3 代。倒置相差显微镜观察各代 NPCs 形态特征;膜联蛋白 Ⅴ/碘化丙啶双染流式细胞术检测细胞凋亡率;免疫细胞荧光染色法和 Western blot 检测各代 NPCs 的缺氧诱导因子 1α(hypoxia-inducible factor 1α,HIF-1α)、聚集蛋白多糖(Aggrecan)、Ⅱ 型胶原、基质金属蛋白酶 2(matrix metalloproteinases 2,MMP-2)表达。结果NPCs 形态从 P0 代的三角形、多角形、多边形逐渐向 P3 代的梭形变化,细胞体积变大,含特征性空泡细胞逐渐消失,细胞生长倍增时间延长。P0~P3 代兔 NPCs 细胞凋亡率分别为 5.47%±0.91%、13.77%±2.42%、33.46%±1.82%、38.76%±1.50%,随着培养代数增加,细胞凋亡率明显增加,比较差异均有统计学意义(P<0.05)。免疫细胞荧光染色示,随着培养代数的增加,HIF-1α、Ⅱ 型胶原和 Aggrecan 荧光强度逐渐减小,MMP-2 荧光强度逐渐增加。Western blot 检测示,HIF-1α 蛋白相对表达量在 P0 代较高,P1 代有升高趋势,而后逐渐降低,各代次间比较差异均有统计学意义(P<0.05)。Ⅱ 型胶原蛋白相对表达量从 P0~P3 代逐渐降低,各代次间比较差异均有统计学意义(P<0.05)。Aggrecan 蛋白相对表达量从 P0~P2 代逐渐降低,两两比较差异均有统计学意义(P<0.05);P2 代与 P3 代间比较差异无统计学意义(P>0.05)。MMP-2 蛋白相对表达量在 P3 代有明显升高,除 P0 代与 P2 代间比较差异无统计意义(P>0.05)外,其余各代次间比较差异均有统计学意义(P<0.05)。结论自然传代培养法可成功构建 NPCs 退变模型,退变时细胞形态变化、凋亡增加,细胞合成代谢减慢,分解代谢加快。

ObjectiveTo research the biological characteristics of different generations of rabbit nucleus pulposus cells (NPCs) that were cultured with natural culture and subculture method.MethodsThe thoracolumbar segments of New Zealand white rabbits (6-8 weeks old and weighing 1.5-2.5 kg) were obtained and nucleus pulposus were isolated from disc regions. And NPCs were harvested by enzymatic digestion from nucleus pulposus. Primary NPCs were counted as P0 generation. Then, NPCs were passaged by trypsin and counted as P1, P2, P3 with a totle of 4 generations. P0 to P3 generations NPCs were separately examined by observation of cell morphology and proliferation time, detection of apoptosis rates of cells by flow cytometry, and detection of hypoxia-inducible factor 1α (HIF-1α), matrix metalloproteinases 2 (MMP-2), Aggrecan, and collagen type Ⅱ proteins by immunofluorescence and Western blot.ResultsThe morphology of NPCs transformed from triangular or polygonal in P0 generation to spindle in P3 generation; the characteristic vacuolated cells gradually disappeared; and the cell volume and cell proliferation time increased. The cell apoptosis rates were 5.47%±0.91%, 13.77%±2.42%, 33.46%±1.82%, and 38.76%±1.50% from P0 to P3 generations, with the increase of culture time, and there were significant differences between 4 generations (P<0.05). Immunofluorescence staining showed that with the increase of cells generation, the fluorescence intensity of HIF-1α, collagen type Ⅱ, and Aggrecan decreased, and the fluorescence intensity of MMP-2 increased. Western blot results showed that the relative expression of HIF-1α protein was high in P0 generation, the P1 generation has a rising trend, and then gradually decreased; the differences between generations were significant (P<0.05). The relative expression of collagen type Ⅱ protein decreased from P0 to P3 generations and there were significant differences between generations (P<0.05). The relative expression of Aggrecan protein decreased from P0 to P2 generations and there were significant differences between generations (P<0.05); but no significant difference was found between P2 and P3 generations (P>0.05). The relative expression of MMP-2 protein increased significantly in P3 generation; except that the difference between P0 and P2 generations was not significant (P>0.05), the significant differences were found between the other generations (P<0.05).ConclusionRabbit NPCs degeneration model was successfully established by the natural culture and subculture method. Transforming of NPCs morphology, increasing of cell apoptosis rates, decreasing of anabolism, and increasing of catabolism were presented in NPCs degeneration model.

关键词: 髓核细胞; 细胞退变; 细胞凋亡;

Key words: Nucleus pulposus cells; degeneration; apoptosis; rabbit

引用本文: 陈琪, 石芳芳, 杨曦, 刘立岷, 宋跃明. 不同代次髓核细胞生物学特性的比较研究. 中国修复重建外科杂志, 2018, 32(6): 660-667. doi: 10.7507/1002-1892.201707017 复制

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1. Kadow T, Sowa G, Vo N, et al. Molecular basis of intervertebral disc degeneration and herniations: what are the important translational questions? Clin Orthop Relat Res, 2014, 473(6): 1903-1912.
2. Lively MW. Sports medicine approach to low back pain. South Med J, 2002, 95(6): 642-646.
3. Oksuz E. Prevalence, risk factors, and preference-based health states of low back pain in a Turkish population. Spine (Phila Pa 1976), 2006, 31(25): E968-972.
4. Yang X, Li X. Nucleus pulposus tissue engineering: a brief review. Eur Spine J, 2009, 18(11): 1564-1572.
5. Rajpurohit R, Risbud MV, Ducheyne P, et al. Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2. Cell Tissue Res, 2002, 308(3): 401-407.
6. Risbud MV, Shapiro IM. Notochordal cells in the adult intervertebral disc: new perspective on an old question. Crit Rev Eukaryot Gene Expr, 2011, 21(1): 29-41.
7. Jones P, Gardner L, Menage J, et al. Intervertebral disc cells as competent phagocytes in vitro: implications for cell death in disc degeneration. Arthritis Res Ther, 2008, 10(4): R86.
8. Peng B, Hao J, Hou S, et al. Possible pathogenesis of painful intervertebral disc degeneration. Spine (Phila Pa 1976), 2006, 31(5): 560-566.
9. Johnstone B, Bayliss MT. The large proteoglycans of the human intervertebral disc. Changes in their biosynthesis and structure with age, topography, and pathology. Spine (Phila Pa 1976), 1995, 20(6): 674-684.
10. Haefeli M, Kalberer F, Saegesser D, et al. The course of macroscopic degeneration in the human lumbar intervertebral disc. Spine (Phila Pa 1976), 2006, 31(14): 1522-1531.
11. 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.
12. Yang F, Leung VY, Luk KD, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. J Pathol, 2009, 218(1): 113-121.
13. Hunter CJ, Matyas JR, Duncan NA. Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison. J Anat, 2004, 205(5): 357-362.
14. Hunter CJ, Matyas JR, Duncan NA. The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering. Tissue Eng, 2003, 9(4): 667-677.
15. Ma X, Lin Y, Yang K, et al. Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro. Int J Mol Med, 2015, 36(1): 186-194.
16. 武海军, 银和平, 李树文, 等. 不同代次兔髓核细胞的形态学特征和生物学性状. 中国组织工程研究, 2013, 17(11): 1901-1908.
17. 刘瑞平, 徐南伟. 兔椎间盘髓核细胞体外退变模型的建立. 江苏医药, 2010, 36(3): 299-301.
18. Risbud MV, Izzo MW, Adams CS, et al. An organ culture system for the study of the nucleus pulposus: description of the system and evaluation of the cells. Spine (Phila Pa 1976), 2003, 28(24): 2652-2658.
19. Lee CR, Iatridis JC, Poveda L, et al. In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies. Spine (Phila Pa 1976), 2006, 31(5): 515-522.
20. Le Maitre CL, Fotheringham AP, Freemont AJ, et al. Development of an in vitro model to test the efficacy of novel therapies for IVD degeneration. J Tissue Eng Regen Med, 2009, 3(6): 461-469.
21. Kasra M, Goel V, Martin J, et al. Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells. J Orthop Res, 2003, 21(4): 597-603.
22. MacLean JJ, Lee CR, Alini M, et al. The effects of short-term load duration on anabolic and catabolic gene expression in the rat tail intervertebral disc. J Orthop Res, 2005, 23(5): 1120-1127.
23. Crean JK, Roberts S, Jaffray DC, et al. Matrix metalloproteinases in the human intervertebral disc: role in disc degeneration and scoliosis. Spine (Phila Pa 1976), 1997, 22(24): 2877-2884.
24. Poiraudeau S, Monteiro I, Anract P, et al. Phenotypic characteristics of rabbit intervertebral disc cells. Comparison with cartilage cells from the same animals. Spine (Phila Pa 1976), 1999, 24(9): 837-844.
25. Eyre DR. Biochemistry of the intervertebral disc. Int Rev Connect Tissue Res, 1979, 8: 227-291.
26. Kim YJ, Sah RL, Doong JY, et al. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem, 1988, 174(1): 168-176.
27. Roberts S, Menage J, Duance V, et al. 1991 Volvo Award in basic sciences. Collagen types around the cells of the intervertebral disc and cartilage end plate: an immunolocalization study. Spine (Phila Pa 1976), 1991, 16(9): 1030-1038.
28. Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
29. Le Maitre CL, Pockert A, Buttle DJ, et al. Matrix synthesis and degradation in human intervertebral disc degeneration. Biochem Soc Trans, 2007, 35(Pt 4): 652-655.