壳聚糖/pCDNA3.1（＋）CrmA对软骨细胞基质金属蛋白酶1及其抑制因子表达的影响

Effect of chitosan/pCDNA3.1 （＋） CrmA on matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 expressions of chondrocytes induced by interleukin-1β

目的 探讨壳聚糖/pCDNA3.1（＋）CrmA对IL-1β诱导的软骨细胞MMP-1及其特异性组织抑制因子（TIMP）-1 mRNA和蛋白表达的影响.方法 体外培养兔关节软骨细胞,分别加入PBS、10μg/ml壳聚糖/pCDNA3.1（＋）和壳聚糖/pCDNA3.1 （＋）CrmA处理后,加入10 ng/ml IL-1β共培养,采用实时定量RT-PCR方法检测各组软骨细胞MMP-1和TIMP-1 mRNA的表达,蛋白印迹法分析各组软骨细胞MMP-1和TIMP-1蛋白的表达.采用单因素方差分析进行统计学分析.结果 壳聚糖/pCDNA3.1 （＋）CrmA处理组软骨细胞MMP-1 mRNA（0.44±0.04）的表达明显低于PBS组（1.00±0.05）和壳聚糖/pCDNA3.1（＋）组（0.76±0.07）,3组比较差异有统计学意义（F=106.93,P＜0.01）.与PBS组（0.73±0.06）和壳聚糖/pCDNA3.1（＋）组（0.46±0.05）比较,壳聚糖/pCDNA3.1 （＋）CrmA处理组（0.28±0.03） MMP-1蛋白的表达显著降低（F=59.66,P＜0.01）.各组间TIMP-1 mRNA和蛋白的表达差异无统计学意义.结论 壳聚糖/pCDNA3.1 （＋）CrmA能够明显抑制IL-1β诱导的软骨细胞MMP-1 mRNA和蛋白的表达,上调IL-1β诱导的软骨细胞TIMPs/MMPs的比值,这可能成为壳聚糖/pCDNA3.1 （＋）CrmA减轻OA软骨退变的原因之一.

Objective To investigate the effects of chitosan/pCDNA3.1 （＋） CrmA on matrix metalloproteinase （MMP）-1 and tissue inhibitor of metalloproteinase （TIMP）-1 expression of chondrocytes induced by interleukin-1β （IL-1β） in mRNA and protein levels.Methods Rabbit chondrocytes were isolated and cultured.Chondrocytes were treated with PBS,10 μg/ml chitosan （CS） and chitosan/ pCDNA3.1 （＋）CrmA respectively for 6 hours.Then 10 ng/ml IL-1β was added into the culture medium.After 48 hours,real time polymerase chain reaction （real time PCR） and Western blot assay were used to examine the changes of MMP1 and TIMP-1 in mRNA and protein levels.Results In CS/pCDNA3.1 （＋）CrmA treated group （0.44±0.04）,the messenger RNA expression of MMP-1 in chondrocytes was significantly suppressed compared with corresponding samples of PBS treated group （1.00±0.05） and CS treated group （0.76±0.07）,There was significant difference between three groups （F=106.93,P〈0.01）.The MMP-1 protein expression of chondrocytes in CS/pCDNA3.1 （＋）CrmA treated group （0.28±0.03） was lower than that of PBS treated group （0.73±0.06） and CS treated group （0.46±0.05）（F=59.66,P〈0.01）.No significant difference of TIMP-1 expression among the three groups was observed in mRNA and protein levels.Conclusion CS/pCDNA3.1（＋） CrmA can significantly inhibit the mRNA and protein expressions of MMP-1 of chondrocytes induced by IL-1β,which leads to up-regulation the ratio of TIMPs to MMPs of IL-1β induced chondrocytes.It may be a part of the mechanisms of the therapeutic effects of CS/pCDNA3.1 （＋）CrmA on osteoarthritis.