CD147抗体对破骨细胞中基质金属蛋白酶活性影响的体外研究

目的:建立人破骨细胞(Osteoclast,OCs)体外诱导分化模型,研究CD147单克隆抗体对OCs分化过程中基质金属蛋白酶-9(Matrix metalloproteinases 9,MMP-9)和基质金属蛋白酶-2(Matrix metalloproteinases 2,MMP-2)表达及活性的影响。方法:通过采集健康成年志愿者外周血所分离的单个核细胞贴壁培养,应用NF-κB配体激活因子(Receptor or Activator ofNF-KB Ligand,RANKL)与巨噬细胞集落刺激因子(Macrophage colony stimulating factor,M-CSF)诱导单个核细胞向OCs分化。本实验分为抗体组(RANKL+M-CSF+CD147单克隆抗体)与对照组(RANKL+M-CSF)。抗酒石酸酸性磷酸酶染色(Tartrate-resistant acid phosphatase,TRAP)与骨吸收实验检测鉴定OCs分化及活性情况,Real-Time PCR技术检测CD147、MMP-9和MMP-2 mRNA在破骨前体细胞(Osteoclast precursor cells,OPCs)中表达情况,明胶酶谱法检测细胞培养上清中MMP-9、MMP-2酶蛋白的活性变化情况。结果:①对照组经TRAP染色和骨吸收实验检测到破骨样细胞形成并具有骨吸收功能,而抗体组OCs分化及活性均受到抑制;②在24、48小时时相点上抗体组CD147、MMP-2及MMP-9 mRNA的相对表达量均低于相应的对照组(P<0.05),且MMP-2、MMP-9 mRNA的相对表达量与CD147 mRNA的表达量呈正相关。③明胶酶谱检测细胞培养上清,可见在24、48小时两时相点上抗体组OCs细胞中MMP-2、MMP-9酶原及活性酶的酶解量均较相应对照组明显降低(P<0.05)。结论:CD147单克隆抗体可以抑制OCs分化成熟过程中MMP-9及MMP-2的表达与活性;CD147对MMP-2、MMP-9活性的调节,可能是其对OCs活化调节的机制之一。

Effect of CD147 monoclonal antibody on activity of matrix metalloproteinase on differentiation of osteoclast in vitro

Objective:To establish the model of differentiation and maturation of osteoclast(Ocs) in vitro and to studies the effect of CD147 monoclonal antibody on matrix metalloproteinases-2,9 expressed in differentiation of osteoclast in vitro.Methods:OCs were induced by peripheral blood mononuclear cells(PBMCs)of human using medium with macrophage colony stimulating factor(M-CSF) and receptor activator of nuclear factor-kappaB ligand(RANKL).PBMCs were divided into 2 groups,antibody group(RANKL+M-CSF+CD147Ab) and control group(RANKL+M-CSF).The maturation of osteoclasts was observed by tartrate-resistant acid phosphatase(TRAP) staining and the function of osteoclasts was analyzed by bone resorption test.The mRNA levels of CD147,MMP-2 and MMP-9 were detected by Real-Time PCR.And the activity of MMP-2,MMP-9 expressed by osteoclast was assessed by zymography analyses.Results:①The formation of osteoclast-like cells and bone resorption can be detected by TRAP staining and bone resorption test in control group,while the formation and activity of OCs were inhibited in antibody group.②The mRNA levels of CD147,MMP-2 and MMP-9 of the antibody group at 24 h,48 h time points were lower than the corresponding control group(P<0.05).Moreover,the mRNA levels of MMP-2,MMP-9 were significantly positively correlated to the mRNA levels of CD147.③The digestion capacity of MMP-2,MMP-9 zymogen and active enzyme of the antibody group at 24 h,48 h time points were lower than the corresponding control group′s(P<0.05) by Gelatin zymography analyses.Conclusion:CD147 monoclonal antibody can inhibit the expression and activity of MMP-2 and MMP-9 in the differentiation and maturation of osteoclast.One of the possible mechanisms activation of OCs regulated by CD147 may be achieved by regulating the expression and activity of MMP-2 and MMP-9.