中性粒细胞增强类风湿关节炎滑膜细胞产生基质金属蛋白酶及其细胞侵袭力

目的：研究中性粒细胞表面CD147分子对类风湿关节炎（RA）成纤维样滑膜细胞（FLS）产生基质金属蛋白酶（MMP）及细胞侵袭力的作用。方法：取自关节镜或骨科手术治疗的RA、骨关节炎（OA）患者的滑膜组织，体外分离培养FLS，通过流式细胞术（FCM）检测细胞表面分子及形态学方法鉴定。采用全反式维甲酸（ATRA）诱导HL-60细胞（人早幼粒白血病细胞株）诱导中性粒细胞分化，并用硝基蓝四氮唑（NBT）还原反应测定其分化程度。进而以FCM测定分化前后的HL-60细胞及FLS表面CD147的表达，并通过明胶酶谱和重组基质侵袭实验检测分化前后HL-60细胞对FLS MMP表达和细胞侵袭能力的作用以及CD147拮抗肽（AP-9）对这种MMP表达和细胞侵袭能力的抑制作用。结果：成功建立了FLS分离培养和HL-60细胞分化的实验体系。培养的FLS具有典型成纤维细胞的形态，均一的高度表达CD90（〉98％）而不表达CD14。分化后的HL-60细胞CD147表达上调，RAFLS细胞CD147表达水平低于分化前及分化后HL-60细胞（P〈0．05）。明胶酶谱试验显示：①与OAFLS相比，单培养的RAFLS表达较高水平的酶原及活化形式的MMP-2（P〈0．05）；②分化前／后的HL-60细胞分别与FLS共培养，酶原及活化形式的MMP-2和MMP-9的表达水平较这些细胞单独培养均显著升高（P〈0．05），且分化后的HL-60细胞与FLS共培养酶原及活化形式MMP-2表达水平较分化前HL-60细胞与FLS共培养明显升高（P〈0．05）；③AP-9显著抑制两共培养组中MMP-2和MMP-9的上调作用（P〈0．05）。侵袭实验显示：RAFLS侵袭能力高于OA FLS（P〈0．05）；分化前后的HL-60均可增加RAFLS的侵袭能力（P〈0．05），且分化后强于分化前（P〈0．05）；AP-9可抑制这一促进作用，降低FLS的侵袭能力（P〈0．05）。结论：中性粒细胞可能通过CD147促进RA患者滑膜FLS MMP-2、MMP-9的表达和活化，增强FLS的侵袭能力，这种促进作用可被CD147拈抗肽所抑制，将为RA关节软骨和骨损伤机制和治疗的进一步研究提供了重要的线索。

Neutrophil enhanced matrix metalloproteinase production and invasion of synoviocytes of RA via CD147

AIM: To study the effects of CD147 on neutrophil on matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) production by fibroblast-like synoviocytes (FLS) and the invasive potential of FLS in rheumatoid arthritis(RA). METHODS: FLS were isolated from the synovial tissues which were resected from the patients with rheumatoid arthritis (RA) and ostarthritis (OA) during synovectomy, and CD14, CD90 on RA FLS were detected by FCM. HL-60 cells were differentiated into mature neutrophil by alltransretinoic acid (ATRA) and the degree of cell differentiation was detected through NBT reduction reaction. CD147 expression on the differentiated and undifferentiated HL-60 cells and FLS were detected by FCM. The release and activity of MMP-2 and MMP-9 were detected in the supernantents of HL-60 cells cocultured with RA FLS by Gelatin zymography. The invasive potential of RA FLS was detected by invasion assay. To further investigate the effect of CD147 on neutrophil on MMP production by FLS and the invasive potential of FLS in RA, antagonist peptide against EMMPRIN/CD147 (AP-9) was added to the coculture. RESULTS: The experimental system of FLS separation and culture and HL-60 differentiation were established successfully. The isolated FLS of RA were characterized by negative CD14 and positive CD90. CD147 expression on the differentiated HL-60 cells was higher than that on the undifferentiated HL-60 cells, and CD147 expression on both of them were higher than that on RA FLS (P < 0.05). The expressions of Pro-MMP-2, MMP-2 produced by RA FLS were higher than that produced by OA FLS (P < 0.05). Pro-MMP-2, MMP-2, Pro-MMP-9 and MMP-9 were significantly increased when RA FLS were cocultured with the undifferentiated or differentiated HL-60 cells (P < 0.05), and Pro-MMP-2 and MMP-2 in differentiated HL-60 cells cocultured with RA FLS increased more than those in the undifferentiated HL-60 cells cocultured with RA FLS (P < 0.05). The increase was inhibited by AP-9 significantly (P < 0.05). Invasion assay showed that the invasive potential of RA FLS was higher than that of OA FLS (P < 0.05), and the undifferentiated or differentiated HL-60 cells increased the invasive potential of RA FLS (P < 0.05), which could be blocked by AP-9. CONCLUSION: Neutrophil might increase the production of MMP-2 and MMP-9 and enhanced the invasion of RA FLS via CD147, which can be inhibited by HAb18G/CD147 antagonistic peptide (AP9). Our research on RA pathogenesis and the mechanism of cartilage destruction may give us some good ideas for RA therapy in future.