CD137-CD137配体对巨噬细胞基质金属蛋白酶9及血管平滑肌细胞钙化形成的影响

Effect of CD137-CD137L signals on macrophage-derived MMP-9 and calcification of vascular smooth muscle cells

目的探讨CD137-CD137配体(CD137L)信号是否通过调控巨噬细胞基质金属蛋白酶9(MMP-9)的表达影响血管平滑肌细胞(VSMC)钙化形成。方法采用巯基乙酸盐腹腔灌洗法获取C57BL/6J小鼠腹腔巨噬细胞(PM),采用组织块贴壁法取3~6代胸主动脉VSMC分为对照组、共培养组、CD137激动组和MMP-9抑制组(n=3)。对照组用重组CD137L蛋白干预VSMC,共培养组用PM和VSMC共培养,CD137激动组和MMP-9抑制组分别用重组CD137L蛋白和MMP-9抑制剂干预PM后,与VSMC共培养,再加入重组CD137蛋白(10μg/ml)干预。采用Western blot检测各组钙化相关的骨桥蛋白(OPN)、Runt相关转录因子2(Runx-2)表达,微板法检测钙离子浓度和碱性磷酸酶(ALP)活性,Von Kossa和茜素红染色检测各组细胞钙化程度。结果CD137激动组VSMC中OPN和Runx-2蛋白表达、钙离子浓度及ALP活性明显高于共培养组(P<0.01),而MMP-9抑制组VSMC中OPN和Runx-2蛋白表达、钙离子浓度及ALP活性明显低于CD137激动组,差异有统计学意义[0.25±0.27 vs 2.12±0.34,0.30±0.32 vs 2.63±0.41,(1.92±0.09)mmol/g vs(4.99±0.37)mmol/g,(1.11±0.50)KU/g vs(2.63±0.04)KU/g,P<0.01]。共培养组VSMC中OPN和Runx-2蛋白表达、钙离子浓度及ALP活性与对照组比较,无统计学意义(P>0.05)。CD137激动组茜素红染色红色颗粒物质沉积和Von Kossa染色黑色颗粒物质沉积明显多于对照组和共培养组;MMP-9抑制组钙化程度显著降低;对照组和共培养组钙化程度则无明显差异。结论CD137-CD137L信号可能通过调控巨噬细胞MMP-9表达影响VSMC钙化形成。

Objective To study whether CD13-CD137L signals can affect the calcification of VSMC by regulating the expression of macrophage-derived MMP-9.Methods Peritoneal macrophages(PM)were isolated from C57BL/6J mice by thioglycolate peritoneal lavage.The VSMC isolated from mouse thoracic aorta were primarily cultured with tissue block adherence method and divided into control group,co-culture group,CD137 activation group and MMP-9 inhibition group.The VSMC in 4 groups were treated with recombinant CD137L,PM,recombinant CD137L protein and MMP-9 inhibitors respectively.The expressions of calcification-related osteopontin(OPN)and Runx-2 were detected by Western blot.The calcium ion concentration and ALP activity were measured by quantitative colorimetry.The calcification of VSMC was assessed with Von Kossa staining and alizarin red staining respectively.Results The OPN and Runx-2 protein expression levels,calcium ion concentration and ALP activity were significantly higher in CD137 activation group than in co-culture group(P<0.01)while they were significantly lower in MMP-9 inhibition group than in CD137 activation group(0.25±0.27 vs 2.12±0.34,0.30±0.32 vs 2.63±0.41,1.92±0.09 mmol/g vs 4.99±0.37 mmol/g,1.11±0.50 KU/g vs 2.63±0.04 KU/g,P<0.01).No significant difference was detected in OPN and Runx-2 protein expression,calcium ion concentration and ALP activity between co-culture group and control group(P>0.05).The number of deposited red particles detected with alizarin red staining and that of deposited black particles detected with Von Kossa staining were significantly greater in CD137 activation group than in control group and co-culture group(P<0.01).The calcification level of VSMC was significantly lower in MMP-9 inhibition group than in control group and co-culture group(P<0.01).No significant difference was detected in calcification level of VSMC between control group and co-culture group(P>0.05).Conclusion CD137-CD137L signals can affect the calcification of VSMC by upregulating the expression of macrophage-derived MMP-9.