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目的研究膜联蛋白A2(ANXA2)磷酸化修饰对类风湿性关节炎成纤维细胞样滑膜细胞(RAFLS)基质金属蛋白酶2(MMP-2)和MMP-9水平及活性的影响。方法原代培养RAFLS,构建包装ANXA2干涉慢病毒,制备ANXA2显著下调的FLS(si ANXA2/FLS)及同型对照作为细胞模型,采用2型胶原蛋白刺激法间接磷酸化RAFLS中的ANXA2。实时定量PCR(qRT-PCR)分析ANXA2磷酸化对RAFLS细胞中MMP-2和MMP-9表达的影响,明胶酶谱实验观察ANXA2活化对RAFLS中MMP-2和MMP-9活性的影响。结果 qRT-PCR结果提示ANXA2磷酸化修饰对RAFLS中MMP-2和MMP-9的水平无显著影响,明胶酶谱实验显示ANXA2磷酸化修饰能够增强MMP-9的活性。结论通过胶原蛋白刺激法间接促进RAFLS中ANXA2的磷酸化修饰,显著增强MMP-9的活性。
Objective To investigate the effect of ANXA2 phosphorylation on the levels and activities of matrix metalloproteinase 2 (MMP-2) and MMP-9 in rheumatoid arthritis fibroblast-like synoviocytes (RAFLS). Methods RAFLS was cultured in primary culture, ANXA2 interference lentivirus was packaged and FLS (si ANXA2 / FLS) and isotype control were downregulated. ANXA2 in RAFLS was indirectly phosphorylated by type 2 collagen stimulation. The effect of ANXA2 phosphorylation on the expression of MMP-2 and MMP-9 in RAFLS cells was analyzed by qRT-PCR. The effect of ANXA2 activation on the activity of MMP-2 and MMP-9 in RAFLS cells was observed by gelatin zymography. Results The results of qRT-PCR suggested that the phosphorylation of ANXA2 had no significant effect on the levels of MMP-2 and MMP-9 in RAFLS. Gelatin zymography showed that the phosphorylation of ANXA2 enhanced the activity of MMP-9. CONCLUSIONS: Indirect stimulation of ANXA2 phosphorylation in RAFLS by collagen stimulation significantly enhances MMP-9 activity.