软骨修复组织蛋白多糖代谢与一氧化氮合酶抑制剂的影响

Effect of nitric oxide synthase inhibitor on proteoglycan metabolism in repaired articular cartilage

目的:应用一氧化氮合酶抑制剂可改善骨性关节炎和风湿性关节炎软骨的代谢,作者前期的实验也证明一氧化氮合酶抑制剂能提高软骨修复组织的质量。实验进一步观察一氧化氮合酶抑制剂对软骨修复组织蛋白多糖代谢的影响。方法:实验于1999-06/2002-02在南方医科大学完成。①实验分组:取雄性新西兰兔24只,8月龄,体质量(2.5±0.2)kg。随机抽签法分为对照组、骨形态发生蛋白组和S-甲基异硫脲组,每组8只。②实验方法:将大白兔双侧股骨髁间关节面造成全层软骨缺损,对照组:软骨缺损不充填任何物质;骨形态发生蛋白组:缺损用骨形态发生蛋白纤维蛋白凝胶复合物充填;S-甲基异硫脲组:缺损应用胶原复合骨形态发生蛋白充填,术后按5mg/(kg·12h)皮下注射S-甲基异硫脲。术后1年麻醉后处死动物。③实验评估:应用组织切片番红O-快绿染色和图像分析技术,按照染色百分率、平均灰度(平均染色程度)和染色厚度(软骨厚度)指标来检测糖胺聚糖含量;应用Na235SO4掺入法检测软骨修复组织蛋白多糖合成。结果:纳入新西兰兔24只,均进入结果分析。①术后1年,对照组几乎无红色染色区域;骨形态发生蛋白组可见到少量的不均匀红色区域;S-甲基异硫脲组可见到较多均匀一致的红色染色区域。②S-甲基异硫脲组软骨修复组织番红O染色百分率为89.28%,明显高于骨形态发生蛋白组36.54%和对照组13.4%,S-甲基异硫脲组修复组织番红O-快绿染色平均灰度值134.5,分别为骨形态发生蛋白组平均灰度值56.8的2.5倍,为对照组26.4的7倍。软骨平均厚度S-甲基异硫脲组1.75cm分别为骨形态发生蛋白组0.76cm和对照组0.25cm的2倍和6倍。③Na235SO4掺入法结果显示,S-甲基异硫脲组[35S]摄入量明显高于骨形态发生蛋白组和对照组(P<0.01)。结论:诱导型一氧化氮合酶抑制剂S-甲基异硫脲的应用能明显增加软骨修复组织糖胺聚糖含量和蛋白多糖合成,对于软骨修复质量的提高有积极意义。

AIM： Nitric oxide synthase inhibitor can improve the chondral metabolism of osteoarthritis and rheumarthritis patients, it is also proved to benefit for repairing the cartilage tissue in the previous study. This study was aimed to further examine the proteoglycan synthesis in repaired cartilage treated with nitdc oxide synthase inhibitor. METHODS： The experiment was carried out in the Southern Medical University from June 1999 to February 2002.① Twenty-four male New Zealand white rabbits, aged 8 months and weighed （2.5±0.2） kg, were divided into control group, bone morphogenetic protein （BMP） group and s-methylisourea （SMT） group, with 8 animals in each group.② Full-thickness defects of cartilage were created in the femoral condyles of rabbits. Sixteen defects in control group were empty, sixteen in BMP group were filled with collagen fibrin glue impregnated with BMP, and sixteen in SMT group were filled with collagen fibrin glue impregnated with BMP and hypodermic injection with SMT （5 mg/kg per 12 hours）. All the animals were killed with anesthesia one-year postoperatively. ③Tissue sections were stained with safranin O-fast green that analyzed by Quantiment 500 image analysis system. Glycosaminoglycan content was assessed by using the following parameters： percentage of safranin O stained area, stain thickness （cartilage thickness） and mean gray scale （average stain intensity）. Incorporation of radiolabelled sodium sulphate Na2^35O4 was used to assess the proteoglycan synthesis. RESULTS Totally 24 rabbits were involved in the result analysis.①One-year postoperatively, no stained area was found in control group; a small amount of tissues stained unevenly appeared in BMP group; a large amount of tissues stained evenly were observed in SMT group.②The percentage of safranin O stained area of repair tissue in SMT group （89.28%） was higher obviouslythan that in BMP group （36.54%） and control group （13.4%）. Mean gray scale in SMT group （134.5） was 2.5 times more than that in BMP group （56.8） and 7 times than the controls （26.4）. Mean cartilage thickness of SMT group （1.75 cm） was 2 times as many as that in BMP group （0.76 cm） and control group （0.25 cm）. ③Na23SO4 incorporation result showed that, the proteoglycan synthesis in the defects treated with SMT was significantly higher than BMP group and control group （P 〈 0.01）. CONCLUSION： Inducible nitric oxide synthase inhibitor SMT can increase glycosaminoglycan content and proteoglycan synthesis, suggesting an active role for ameliorating the cartilage repairs.