摘要
目的:探讨羧甲基壳聚糖抑制重组人白细胞介素-1β(rhIL-1β)刺激下的兔软骨细胞合成基质金属蛋白酶-1,3(MMP-1,3)的作用机制。方法:分离、培养兔软骨细胞,用rhIL-1β刺激,同时加入不同浓度的羧甲基壳聚糖(CM-chitosan)或L-精氨酸甲酯(L-NAME),培养24h后,检测上清液中的一氧化氮含量和软骨细胞内的诱导性一氧化氮合酶(iNOS)活力。提取细胞总RNA,采用逆转录聚合酶链方法(RT-PCR)检测iNOS,MMP-1,MMP-3的mRNA表达。通过ELISA方法检测上清液中MMP-1,MMP-3的含量。结果:IL-1β能够增加软骨细胞一氧化氮的释放及iNOS的酶活性,诱导细胞iNOS,MMP-1,3的mRNA表达,增加MMP-1,3的分泌。羧甲基壳聚糖对软骨细胞一氧化氮的释放,iNOS酶活力及iNOSmRNA表达均有抑制作用,不同浓度的抑制效果相近;它对软骨细胞的MMP-1,3mRNA的表达及MMP-1,3的分泌也有明显的抑制作用,且呈剂量依赖性。L-NAME对软骨细胞的MMP-1,3mRNA的表达及分泌也有抑制作用,但L-NAME与羧甲基壳聚糖对一氧化氮的影响相似时,羧甲基壳聚糖对MMP-1,3mRNA表达和分泌的抑制作用强于L-NAME。结论:羧甲基壳聚糖抑制软骨细胞合成MMP-1,MMP-3是部分通过降低iNOS表达,减少一氧化氮合成来实现的。
Objective：To study the mechanism that carboxymethyl-chitosan（CM-chitosan）inhibits the matrix metalloproteinase-1and-3（MMP-1and-3）expression of recombinant human interleukin-1β（rhIL-1β）-induced rabbit chondrocytes.Methods：Chondrocytes were isolated and cultured.The rhIL-1βat a concentration of 10μg/L with CM-chitosan of varied concentrations or L-NAME was added into culture medium.After 24hours,nitric oxide（NO）production and inducible NO synthase（iNOS）activity were detected.The total RNA of chondrocytes were extracted and the MMP-1,-3and iNOS mRNA were examined by semi-quantitative reverse transcription-polymerase chain reaction（RT-PCR）.ELISA was used to detect the concentration of MMP-1,-3in cellcultured fluid.Results：rhIL-1βincreased NO production,iNOS activity,induced the expression of MMP-1,-3and iNOS mRNA and enhanced MMP-1,-3secretion.CM-chitosan could significantly inhibit the production of NO,iNOS activity and iNOS mRNA expression and the inhibitory effects of different concentrations of CM-chitosan were similar;CM-chitosan also decreased significantly the MMP-1,-3mRNA expression and secretion in a dose-dependent manner.L-NAME could inhibit the NO production,MMP-1,-3mRNA expression and secretion,but its inhibitory effect on MMP-1,-3expression and secretion was lower than CM-chitosan when they had the similar inhibitory effects on NO production.Conclusion：The inhibitory effect of CM-chitosan on MMP-1,-3of IL-1β-induced chondrocytes is partly attributed to its ability of suppression of NO production and iNOS expression.
出处
《武汉大学学报（医学版）》
CAS
北大核心
2010年第6期726-730,共5页
Medical Journal of Wuhan University
基金
国家自然科学基金资助项目(编号:30801166)
湖北省科技攻关基金项目(编号:2003AA301C11)
