塞来昔布对钛颗粒诱导破骨细胞活化的抑制作用  

Inhibitory effect of celecoxib on titanium particle-induced osteoclastogenesis through inactivation of cyclooxygenase-2

作　　者：耿德春[1] 杨惠林[1] 朱雪松[1] 毛海青[1] 邹俊[1] 徐耀增[1] 

机构地区：[1]苏州大学附属第一医院骨科,215006

出　　处：《中华实验外科杂志》2013年第10期2161-2164,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81272018、81101399、81101370）;江苏省自然科学基金资助项目（BK2011303）;江苏省科技支撑计划资助项目（BE2011672）;江苏省研究生培养创新工程资助项目

摘　　要：目的 观察环氧合酶-2（COX-2）选择性抑制剂-塞来昔布（Cel）对钛颗粒（Ti）诱导破骨细胞（OC）活化的影响.方法 实验分为6组：空白组、Ti组、核因子（NF）-κB受体活化因子配体（RANKL）组、R＋Ti组、Cel组和前列腺素E2（PGE2）组.采用噻唑蓝（MTT）法检测0.lg/L Ti和不同浓度Cel（10、50、100、200、500μg/L）处理小鼠单核/巨噬细胞株RAW264.7后12、24、48、72 h细胞增殖活性；以抗酒石酸酸性磷酸酶（TRAP）染色检测成熟OC,以逆转录-聚合酶链反应（RT-PCR）检测COX-2、核因子（NF）-κB受体活化因子（RANK）、TRAP、活化T细胞核因子c1（NFATc1）和组织蛋白酶K（CPK）基因mRNA含量,Western blot分析COX-2蛋白表达变化,酶联免疫吸附试验（ELISA）检测PGE2表达水平.结果 MTT结果表明Ti和（或）Cel（≤500 μg/L）对RAW264.7细胞增殖能力无影响.Western blot及RT-PCR结果表明COX-2表达峰值出现在RANKL和Ti作用后2h,24 h后COX-2表达量与空白组比较差异无统计学意义（P＞0.05）.ELISA结果显示,RANKL和Ti加入后12h,PGE2的含量为3.912±0.231,与Cel预处理组（1.488±0.093）比较,差异有统计学意义（P＜0.05）.TRAP染色,RANKL组、R＋Ti组、Cel后处理组和PGE2组均可见大量紫红色多核细胞；Ti组和Cel预处理组阳性细胞较少,统计分析结果表明Cel≥100 μg/L时,TRAP阳性细胞数量与RANKL组比较差异有统计学意义（P＜0.05）.RT-PCR结果显示Cel组（100 μg/L）RANK、TRAP、NFATcl和CPK基因mRNA含量分别为4.980 ±0.180、3.128±0.143、6.166±0.223和4.922±0.163,与R ＋Ti组和PGE2组之间的差异有统计学意义（P＜0.05）.结论 预先给予选择性COX-2抑制剂Cel能减少PGE2分泌,抑制RANK的表达,阻断Ti诱导的OC活化.Objective To explore the effect of celecoxib,cyclooxygenase （COX）-2 selective inhibitor,on the regulation of osteoclast differentiation from a murine macrophage cell line （RAW264.7） stimulated with titanium （Ti） particles and the receptor activator of nuclear factor-κB （NF-κB） ligand （RANKL）.Methods The effect of celecoxib and Ti particles on RAW264.7 cell viability was examined using methyl thiazol tetrazolium （MTT） assay.Osteoclast formation was measured by tartrate resistant acid phosphatase （TRAP） staining.The mRNA expression levels of COX-2,NF-κB receptor activation factor （RANK）,TRAP,nuclear factor of activated T-cells cytoplasmic 1 （NFATcl） and cathepsin K （CPK） were detected by using quantitative reverse transcription-polymerase chain reaction （RT-PCR）.The COX-2 protein level was determined by using Western blotting.Enzyme-linked immunosorbent assay （ELISA） was performed to determine prostaglandin E2 （PGE2）.Results Celecoxib （10-500 μg/L） did not affect RAW264.7 cell viability.The qRT-PCR results showed that COX-2 gene expression was detectable within 30 min after exposure to Ti particles,and a maximal level was observed by 2 h.Under basal conditions,COX-2 protein expression was undetectable,but the expression was observed within a 1-h exposure to Ti particles.Maximal levels were present by 2 h and 4 h after stimulation with Ti particles.Ti stimulation induced PEG2 production in RAW264.7 cells.Consistently,pretreatment of celecoxib completely inhibited Ti particle-induced PGE2 secretion.However,post-treatment of celecoxib did not affect the PGE2 production.In our study,mature osteoclasts were obtained from RAW264.7 cells stimulated with RANKL and Ti particles for 6 days,which were detected by TRAP staining.Celecoxib doses ≥ 100 μg/L significantly reduced the number of TRAP-positive cells as compared with controls.After 6 days in culture,the mRNA expression levels of RANK,TRAP,NFATc1 and CPK were significantly higher in the presence of RANKL an

关 键 词：无菌性松动 破骨细胞 塞来昔布 环氧合酶-2 前列腺素E2 

分 类 号：R965[医药卫生—药理学]