PDGF-D抗体对体外破骨前体细胞分化过程影响的相关研究

Effect of platelet-derived growth factor-D antibody on differentiation and maturation of osteoclasts in vitro

目的:用外源性血小板衍生生长因子-D抗体(Platelet-derived growth factor D antibody,PDGF-DAb)体外刺激外周血单核细胞(Peripheral blood mononuclear cells,PBMCs),观察其对破骨前体细胞分化过程的影响。方法:采集血并分离单个核细胞,将所得细胞分为三组:诱导组(PDGF-D+PBMCs)、阻断组(PDGF-D+PDGF-D Ab)和对照组(细胞培养基+PBMCs)。于第15天应用抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,TRAP)染色和骨吸收实验观察破骨样细胞(Osteoclastlike cells,OLCs)的分化及活性;Real-time PCR检测OLCs标志基因TRAP、Cathepsin K、MMP-9(Matrix metalloproteinases 9)mR-NA的表达;Western blot检测各组细胞中Cathepsin K蛋白的表达;酶联免疫吸附试验(ELISA)检测各组细胞上清液中MMP-9的表达。结果:TRAP染色和骨吸收实验见诱导组有大量具有骨吸收功能的OLCs,而阻断组和对照组均未见。阻断组和对照组TRAP、Cathepsin K、MMP-9 mRNA的相对表达量均显著低于诱导组(P<0.05),且二组间无显著差异(P>0.05)。对照组和阻断组中Cathepsin K蛋白(Western blot检测)和MMP-9(ELISA检测)表达均低于诱导组(P<0.05),且两组间无显著差异(P>0.05)。结论:外源性PDGF-D抗体可阻断由PDGF-D诱导的PBMCs向OLCs分化过程。

Objective：To study the effects of PDGF-D Ab on the differentiation and maturation of osteoclasts（OCs） in vitro.Methods： Peripheral blood mononuclear cells（PBMCs） were gathered and isolated from the fresh blood,and then they were divided into three groups： The induction group（IG）： PBMCs were induced by using PDGF-D.The control group（CG）：PBMCs were threated with nothing but culture medium.The blocking group（BG）： PBMCs were threated by using PDGF-D and PDGF-D antibody.At day 15,mRNA levels of tartate-resistant acid phosphatase（TRAP）,matrix metalloproteinases 9（MMP-9） and Cathepsin K were detected by Real-time PCR.The differentiation and maturation of OCs were judged by TRAP staining and the function of osteoclasts by bone resorption test.The expression of Cathepsin K were detected by Western blot and the protein expression level of MMP-9 were detected by ELISA.Results： The OC-like cells were detected and showed in TRAP staining test and bone resorption test in IG,but there was no one detected in CG and BG.mRNA levels of TRAP,MMP-9,Cathepsin K at day 15 were higher in IG than those in the CG and BG（P0.05）,and at the same time,there was no different between CG and BG（P0.05）.As the same as the up results,the protein expression levels of MMP-9（ELISA） and Cathepsin K（Western blot） were higher in IG（P0.05）,but there were no diffrences between CG and BG（P0.05）.Conclusion： OC-like cells induced by PDGF-D with PBMCs could be blocked by PDGF-D antibody in vitro.