肿瘤坏死因子-α作用下椎间盘退变动物模型的建立
Establishment of animal model of intervertebral disc degeneration induced by TNF-α
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摘要: 目的:制作家兔椎间盘退变(IVDD)的动物模型,研究肿瘤坏死因子-α(TNF-α)对髓核组织的影响。方法:取健康成年家兔30只,手术暴露L2~L6,L3/4、L4/5作为实验组,每个椎间盘髓核注射20 ng/μL TNF-α 15 μL;L5/6作为对照组,每个椎间盘髓核注射15 μL磷酸缓冲盐溶液;L2/3作为空白组,不做处理。于术后4周、8周、12周行腰椎X线及磁共振(MRI)检查,每次随机检查10只家兔,检查完后处死并取其髓核组织经行免疫组织化学及原位末端标记检查。结果:术后4~12周,X线见实验组椎间隙高度进行性下降,MRI可见实验组椎间隙T2加权像信号逐渐降低;免疫组织化学及TUNEL显示实验组髓核组织内Ⅰ型胶原及细胞凋亡率随之时间推移逐渐增加,Ⅱ型胶原逐渐减少。对照组和空白组差异无统计学意义(P>0.05)。结论:实验成功建立了TNF-α作用下的IVDD动物模型。TNF-α介导的椎间盘术后椎间隙高度丢失,同时TNF-α可诱导椎间盘髓核细胞退变及凋亡。微量注射器的穿刺作用对髓核细胞的退变及凋亡无明显影响。Abstract: Objective:To establish the animal model of intervertebral disc degeneration(IVDD) induced by tumor necrosis factor-α(TNF-α),and investigate the influence of TNF-α on nucleus pulposus tissue.Methods:The nucleus pulposus of intervertebral disc in L2 to L6,L3/4 and L4/5 in 30 New Zealand white rabbits were injected with 15 μL of 20 ng/μL TNF-α,and set as the experimental group.The nucleus pulposus of intervertebral disc in L5/6 was injected with 15 μL of PBS,and set as the control group.The non-treatment L2/3 of rabbit was set as the blank group.Ten rabbits were randomly selected,and detected using X-ray and MRI after 4,8 and 12 weeks of operation,respectively.The nucleus pulposus tissue of rabbits were examined using immunohistochemistry and TUNEL.Results:After 4 to 12 weeks of operation,the height and T2 weighted image signal of intervertebral disc in experimental group gradually decreased under X-ray and MRI,respectively.The results of immunohistochemistry and TUNEL showed that the expression level of typeⅠcollagen and cell apoptosis in experimental group gradually increased with the time extending,the expression level of type Ⅱ collagen gradually decreased,and the differences of which between the control group and blank group was not statistically significant(P>0.05).Conclusions:The IVDD animal model induced by TNF-α is successfully established.The height of intervertebral disc loses under the mediating of TNF-α,and TNF-α can induce the degeneration and apoptosis of nucleus pulposus cells.The microsyringe puncture can not affect the degeneration and apoptosis of nucleus pulposus cells.
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[1] BECKER A,HELD H,REDAELLI M,et al.Low back pain in primary care:costs of care and prediction of future health care utilization[J].Spine (Phila Pa 1976),2010,35(18):1714. [2] DAGENAIS S,CARO J,HALDEMAN S.A systematic review of low back pain cost of illness studies in the United States and internationally[J].Spine J,2008,8(1):8. [3] ASCHE CV,KIRKNESS CS,MCADAM-MARX C,et al.The societal costs of low back pain:data published between 2001 and 2007[J].J Pain Palliat Care Pharmacother,2007,21(4):25. [4] SMITH LJ,CHIARO JA,NERURKAR NL,et al.Nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine challenge following long-term agarose culture[J].Eur Cell Mater,2011(22):291. [5] STUDER RK,VO N,SOWA G,et al.Human nucleus pulposus cells react to IL-6:independent actions and amplification of response to IL-1 and TNF-α[J].Spine (Phila Pa 1976),2011,36(8):593. [6] LEE S,MOON CS,SUL D,et al.Comparison of growth factor and cytokine expression in patients with degenerated disc disease and herniated nucleus pulposus[J].Clin Biochem,2009,42(15):1504. [7] MURAB S,CHAMEETTACHAL S,BHATTACHARJEE M,et al.Matrix-embedded cytokines to simulate osteoarthritis-like cartilage microenvironments[J].Tissue Eng Part A,2013,19(5/6):1733. [8] MILLWARD-SADLER SJ,COSTELLO PW,FREEMONT AJ,et al.Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells:implications for the pathogenesis of intervertebral disc degeneration[J].Arthritis Res Ther,2009,11(3):R65. [9] ANDERSON DG,LI X,BALIAN G.A fibronectin fragment alters the metabolism by rabbit interverbral disc cells in vitro[J].Spine,2005,30(11):1242. [10] 胡宝山,丁悦,李春海,等.新型兔腰椎间盘退变模型的建立[J].中国临床解剖学杂志,2006,24(5):546. [11] HIYAMA A,MOCHIDA J,OMI H,et al.Cross talk between Smad transcription factors and TNF-α in intervertebral disc degeneration[J].Biochem Biophys Res Commun,2008,369(2):679. [12] 谭清实.椎间盘源性腰痛发病机制的研究[D].天津:天津医科大学,2010. [13] HAYASHI S,TAIRA A,INOUE G,et al.TNF-α in nucleus pulposus induces sensory nerve growth:a study of the mechanism of discogenic low back pain using TNF-α-deficient mice[J].Spine (Phila Pa 1976),2008,33(14):1542. [14] WEI A,BRISBY H,CHUNG SA,et al.Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis[J].Spine J,2008,8(3):466. [15] 史红伟.肿瘤坏死因子(TNF-α)诱导大鼠髓核细胞凋亡过程中EMP-1、caspase-3基因表达及意义[D].太原:山西医科大学,2012. [16] 尚琦松,史晨辉,王宗亮.退变椎间盘组织中TNF-α及IL-18的表达及意义[J].农垦医学,2008,30(4):263. -

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