课题背景：课题为国家自然科学基金资助项目(30571890)。骨关节炎最终病理发展的结果是关节软骨的破坏，而国内外众多研究表明部分基质金属蛋白酶参与软骨的破坏。近年发现，在动物体内和体外，高表达的一氧化氮可加速骨关节炎软骨的破坏；那么，一氧化氮合酶抑制剂对人骨关节炎软骨细胞增殖和基质金属蛋白酶表达的影响如何？是否和在动物体内体外的实验结果相同？虽然，也在不少相关文献中，找到一些类似的见解，但是缺乏直接的证据。

相关链接：骨关节炎的致病过程涉及到数以百计的分子，并形成网络级联系统，这些分子之间相互影响，相互作用，通过多种激活途径，产生生物学效应。在这些细胞因子中究竟哪一个或哪一些在病理发展过程中是一个“关键点”；是否可以通过这些“关键点”细胞因子的抗体或受体的抑制剂来缓解病理发展过程。随着蛋白质组学技术的发展和应用，便利了该类问题的解决。

术语解析：xMAP技术：最新开发的liquichip workstation液相蛋白芯片系统就是一种新型的蛋白质研究分析平台。利用该系统，可以对同一样品中多个不同分子同时检测，这种检测技术被称为xMAP技术（flexible Muti-Analyte Profiling）。liquichip workstation有机地整合了有色微球、激光技术、应用流体学、最新的高速数学信号处理器和计算机运算法则。这些造就了它无与伦比的检测特异性、灵敏性及操作性。可以想像通过该技术，可以有效的进行骨关节炎相关生长因子和细胞因子的检测，横向比较其关联性，从而筛选骨关节炎致病关键因子。

摘要
目的：研究表明，白细胞介素1β对鼠肾细胞、心肌细胞和神经上皮细胞发挥抗增殖作用，但其是否可抑制人的软骨细胞增殖？这种抗增殖作用是否可被一氧化氮合酶抑制剂抑制尚不清楚。实验拟验证一氧化氮合酶抑制剂对人骨关节炎软骨细胞增殖及基质金属蛋白酶的影响。
方法：选择2006-05/12在南方医科大学附属南方医院脊柱骨病科住院的膝骨关节炎患者8例。所有患者均符合1986年美国风湿病学会关于骨关节炎的临床诊断标准或临床及放射学诊断标准，排除其他疾病（创伤性、风湿性及感染性）所导致的骨关节炎。所有患者均在术前告知，并签有试验知情同意书。分离培养关节软骨细胞，将不同浓度（0，1，10，100 μg/L，其中0 μg/L 作为对照）白细胞介素1β作用于骨关节炎软骨细胞24，48，72 h，一氧化氮合酶抑制剂氨基胍作为干扰因素，与白细胞介素1β共同作用72 h，收集细胞上清液，一氧化氮的表达量通过一氧化氮试剂盒检测，并作为检测一氧化氮合酶抑制剂活性的手段；四甲基偶氮唑盐法检测软骨细胞的增殖；xMAP技术检测软骨细胞基质金属蛋白酶1，基质金属蛋白酶3和基质金属蛋白酶9的表达。
结果：①不同时间点1，10，100 μg/L 白细胞介素1β组作用后的吸光度值与对照组相比，差异显著（P < 0.05，P < 0.01）；相同浓度白细胞介素1β在24，72 h 的吸光度值相比较，差异显著(P < 0.01)。②白细胞介素1β和氨基胍(100 μmol/L)共同作用软骨细胞72 h后，各浓度组吸光度与白细胞介素1β组相比，差异显著（P < 0.01）。③白细胞介素1β可促进软骨细胞一氧化氮的表达，并呈剂量依赖关系。④白细胞介素1β促进基质金属蛋白酶1和基质金属蛋白酶3的表达，与对照组相比，差异有显著性意义（P < 0.01），而软骨细胞基质金属蛋白酶9的表达在本实验中没有检测到。
结论：白细胞介素1β可抑制软骨细胞增殖，诱导基质金属蛋白酶表达，这些影响可被氨基胍抑制，白细胞介素1β和氨基胍对软骨细胞的影响可能是通过一氧化氮途径发挥作用。
关键词：软骨细胞；骨关节炎；白细胞介素1β；一氧化氮合酶抑制剂；一氧化氮；液相蛋白芯片

梁鹏展，江建明，刘传芳，孙玮，余磊，邱小忠. 一氧化氮合酶抑制剂对白细胞介素1β诱导软骨细胞增殖和基质金属蛋白酶表达的影响[J].中国组织工程研究与临床康复，2008，12(15):2820-2824
[www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2820(ps).pdf]

Effects of nitricoxide synthase inhibitor on interleukin-1 beta-induced chondrocyte proliferation and matrix metalloproteinase expression

Abstract

AIM:Studies have demonstrated that interleukin-1β (IL-1β) can inhibit the proliferation of rat renal cells, myocardial cells, and neuroepithelial cells. But whether it can inhibit human chondrocyte proliferation, and whether this inhibitory effect can be suppressed by nitricoxide synthase (NOS) are still uncertain. This study explored the effects of NOS inhibitor on chondrocyte proliferation and matrix metalloproteinase (MMP) in patients with osteoarthritis.
METHODS: Eight inpatients with osteoarthritis were selected from Department of Orthopaedics and Spine Surgery, Nanfang Hospital, Southern Medical University from May to December 2006. All subjects fulfilled the diagnosis criteria for osteoarthritis according to the diagnostic standards by American College of Rheumatology or the clinical and radiological diagnostic standards. Osteoarthritis induced by traumatic, rheumatic or infectious diseases was excluded. The informed consent was obtained from all subjects. After cell isolation and culture, chondrocytes were stimulated with different concentrations (0, 1, 10, 100 μg/L, 0 as control) IL-1β for 24, 48, and 72 hours, separately and treated with aminoguanidine (AG), a kind of NOS inhibitor, for 72 hours. Nitric oxide (NO) was detected using NO kit to identify the activity of NOS inhibitor. Cell proliferation was examined by MTT array, and MMP-1, MMP-3, and MMP-9 production in supernatants were measured by technique of flexible Multi-Analyte Profiling (xMAP).
RESULTS: ①At different time points, there were significant differences in absorbance values between IL-1β groups (1, 10, 100 μg/L) and control group (P < 0.05, P < 0.01); the absorbance values of the same concentration of IL-1β were significantly different at 24 and 72 hours of stimulation (P < 0.01). ②The absorbance values of chondrocytes treated with IL-1β and AG for 72 hours were statistically significant compared with cells only stimulated with IL-1β (P < 0.01). ③IL-1β promoted NO expression in a dose-dependent manner. ④ IL-1βsignificantly stimulated chondrocytes to secret MMP-1, and MMP-3 compared with control group ( P < 0.01), but MMP-9 was not detected in this study.
CONCLUSION: IL-1β could reduce chondrocyte proliferation and induce MMP secretion, but these effects are blocked by AG. IL-1β and AG influence chondrocytes by NO.

Liang PZ, Jiang JM, Liu CF, Sun W, Yu L, Qiu XZ. Effects of nitricoxide synthase inhibitor on interleukin-1 beta-induced chondrocyte proliferation and matrix metalloproteinase expression.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(15):2820-2824(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2820(ps).pdf]