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目的建立体外破骨细胞(osteoclasts,OCs)诱导分化模型,研究CD147对体外破骨细胞分化过程中基质金属蛋白酶-2(matrix metalloproteinases 2,MMP-2)和基质金属蛋白酶-9(matrix metalloproteinases 9,MMP-9)合成与分泌的影响。方法采用健康成人外周血分离单个核细胞贴壁培养,应用巨噬细胞集落刺激因子(macrophage colony stimulatingfactor,M-CSF)与NF-κB受体活化因子配体(receptor or activator of NF-κB ligand,RANKL)诱导单个核细胞向Ocs分化。实验分为对照组(RANKL+M-CSF)与蛋白诱导组(RANKL+M-CSF+CD147蛋白)。应用抗酒石酸酸性磷酸酶染色(tar-trate-resistant acid phosphatase,TRAP)及骨吸收实验检测鉴定OCs的分化成熟,Real-time PCR技术检测CD147、MMP-2和MMP-9 mRNA在破骨前体细胞(osteoclast precursor cells,OPCs)中表达变化,酶联免疫吸附实验(ELISA)检测细胞上清液中MMP-2、MMP-9的表达。结果①TRAP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,建立体外Ocs诱导分化模型成功。②在24、48 h蛋白诱导组CD147 mRNA[(1.344±0.207)、(1.816±0.222)]、MMP-2 mRNA[(1.187±0.077)、(1.437±0.231)]及MMP-9 mRNA[(1.128±0.107)、(2.353±0.679)]的相对表达量均高于相应的对照组(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(r分别为0.818、0.758,P<0.01)。③24、48 h蛋白诱导组MMP-2蛋白[(386.71±40.66)、(367.35±20.72)μg/L]、MMP-9蛋白[(1 177.78±66.42)、(1 514.37±68.56)μg/L]表达均高于相应对照组(P<0.05),蛋白诱导组MMP-2蛋白的表达在24 h与48 h间无统计学差异(t=1.190,P>0.05),而MMP-9蛋白的表达在48 h显著高于24 h(t=7.589,P<0.05)。结论外源性CD147蛋白可以促进OCs分化成熟过程中MMP-2及MMP-9的合成与分泌,但MMP-2分泌的增加不呈时间依赖性。
OBJECTIVE: To establish a model of osteoclasts (OCs) differentiation in vitro and to study the effect of CD147 on the expression of matrix metalloproteinases 2 (MMP-2) and matrix metalloproteinases 9 MMP-9) synthesis and secretion. Methods Human peripheral blood mononuclear cells (PBMCs) were cultured in adherent cells of healthy adults, and the effects of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand RANKL) induces differentiation of mononuclear cells to Ocs. The experiment was divided into control group (RANKL + M-CSF) and protein induction group (RANKL + M-CSF + CD147 protein). The differentiation and maturation of OCs were detected by using TRAP and bone resorption assay. The expressions of CD147, MMP-2 and MMP-9 mRNA in osteoclast precursors (OPCs). The expression of MMP-2 and MMP-9 in the cell supernatant was detected by enzyme linked immunosorbent assay (ELISA). Results ① TRAP staining and bone resorption assay detected osteoclast-like cells with bone resorption function, and established the Ocs-induced differentiation model in vitro successfully. ② The expression of CD147 mRNA [(1.344 ± 0.207), (1.816 ± 0.222)], MMP-2 mRNA [(1.187 ± 0.077), (1.437 ± 0.231)] and MMP-9 mRNA [(1.128 ± 0.107), (2.353 ± 0.679)] were higher than the corresponding control group (P <0.05), and the relative expression of MMP-2 and MMP-9 mRNA was positively correlated with the expression of CD147 mRNA Respectively 0.818,0.758, P <0.01). The protein expressions of MMP-2, MMP-2 and MMP-2 in 24,48 h group were significantly higher than those in control group (P <0.05) (P <0.05). The expression of MMP-2 protein in protein-induced group was not significantly different between 24 h and 48 h (t = 1.190, P> 0.05) 48 h significantly higher than 24 h (t = 7.589, P <0.05). Conclusion Exogenous CD147 protein can promote the synthesis and secretion of MMP-2 and MMP-9 in the process of OCs differentiation and maturation, but the increase of MMP-2 secretion is not time-dependent.