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

摘要

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 phos-phatase (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.%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 of NF-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(Tartrateresistant 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.