Induction of matrix metalloproteinase-2 and-3 activity in ovine nucleus pulposus cells grown in three-dimensional agarose gel culture by interleukin-lβ:: a potential pathway of disc degeneration
Induction of matrix metalloproteinase-2 and-3 activity in ovine nucleus pulposus cells grown in three-dimensional agarose gel culture by interleukin-lβ:: a potential pathway of disc degeneration
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DOI：
10.1007/s00586-002-0454-2
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发表时间：
2003-02-01
影响因子：
2.8
通讯作者：
Taylor, TKF
中科院分区：
文献类型：
--
作者：
Shen, B;Melrose, J;Taylor, TKF
腰椎间盘退变是一个重要的临床问题，经常导致腰痛。由于大约80-90%的普通人群在其一生中的某个阶段会遭受背痛，这具有重大的社会经济后果。基质金属蛋白酶（MMPs）与椎间盘退变过程中细胞外基质成分的过度分解有关。本研究的目的是评价白细胞介素-1 β（IL-1 β）对培养的绵羊髓核细胞（NP）产生MMP-2（明胶酶-A）和MMP-3（基质溶解素）的调节作用。在三维琼脂糖培养中建立NP细胞，并在无血清条件下用IL-1 β刺激。通过明胶和酪蛋白酶谱法以及使用MMP特异性底物的荧光测定法对条件培养基样品进行评价。通过NP细胞对IL-1 β的反应，建立了MMP-2、MMP-3产生的时间过程和剂量依赖性。明胶和酪蛋白酶谱分析表明，培养基样品中存在对IL-1 β处理作出反应的MMP-2和MMP-3原水平升高。培养24-96 h后，活性43和45 kDa活性MMP-3的水平显著升高，而MMP-2主要以其72 kDa前体形式存在。在与IL-1 β长时间孵育后，还存在额外的36、28和21 kDa MMP物质，可能代表MMP分解物质。IL-1 β是NP细胞的有效分解代谢介质，导致培养物中MMP-2和-3水平升高。然而，接近70%的MMP-2以72 kDa的前体形式存在，这表明在其体内活化中涉及一些额外的步骤。
Degeneration of the intervertebral disc is an important clinical problem, which often contributes to low back pain. Since approximately 80-90% of the general population will be subject to back pain at some stage during their lifetime, this has major socioeconomic consequences. Matrix metalloproteinases (MMPs) have been implicated in the excessive breakdown of extracellular matrix components during disc degeneration. The aim of the present study was to evaluate the regulation of MMP-2 (gelatinase-A) and MMP-3 (stromelysin) produced by cultured ovine nucleus pulposus (NP) cells stimulated with interleukin-1beta (IL-1beta). NP cells were established in three-dimensional agarose culture and stimulated with IL-1beta under serum-free conditions. Conditioned media samples were evaluated by gelatin and casein zymography and by fluorimetry using an MMP-specific substrate. Time-course and dose dependencies were established for MMP-2, -3 production by the NP cells in response to the IL-1beta. Gelatin and casein zymography indicated that elevated levels of proMMP-2 and proMMP-3 were present in media samples in response to the IL-1beta treatment. After 24-96 h culture, levels of the active 43 and 45 kDa active MMP-3 were significantly elevated, whereas MMP-2 was present mainly as its 72 kDa pro-form. Additional 36, 28 and 21 kDa MMP species were also present after prolonged incubation with IL-1beta, probably representing MMP breakdown species. IL-1beta was a potent catabolic mediator for the NP cells, resulting in the production of elevated levels of MMP-2 and -3 in culture. However, similar to70% of the MMP-2 was present as the 72 kDa pro-form, which suggests that some additional steps are involved in its activation in vivo.