CAJ | 학술논문

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.
목적 관찰배양합매-2(COX-2)선택성억제제-새래석포(Cel)대태과립(Ti)유도파골세포(OC)활화적영향.방법 실험분위6조:공백조、Ti조、핵인자(NF)-κB수체활화인자배체(RANKL)조、R+Ti조、Cel조화전렬선소E2(PGE2)조.채용새서람(MTT)법검측0.lg/L Ti화불동농도Cel(10、50、100、200、500μg/L)처리소서단핵/거서세포주RAW264.7후12、24、48、72 h세포증식활성；이항주석산산성린산매(TRAP)염색검측성숙OC,이역전록-취합매련반응(RT-PCR)검측COX-2、핵인자(NF)-κB수체활화인자(RANK)、TRAP、활화T세포핵인자c1(NFATc1)화조직단백매K(CPK)기인mRNA함량,Western blot분석COX-2단백표체변화,매련면역흡부시험(ELISA)검측PGE2표체수평.결과 MTT결과표명Ti화(혹)Cel(≤500 μg/L)대RAW264.7세포증식능력무영향.Western blot급RT-PCR결과표명COX-2표체봉치출현재RANKL화Ti작용후2h,24 h후COX-2표체량여공백조비교차이무통계학의의(P＞0.05).ELISA결과현시,RANKL화Ti가입후12h,PGE2적함량위3.912±0.231,여Cel예처리조(1.488±0.093)비교,차이유통계학의의(P＜0.05).TRAP염색,RANKL조、R+Ti조、Cel후처리조화PGE2조균가견대량자홍색다핵세포；Ti조화Cel예처리조양성세포교소,통계분석결과표명Cel≥100 μg/L시,TRAP양성세포수량여RANKL조비교차이유통계학의의(P＜0.05).RT-PCR결과현시Cel조(100 μg/L)RANK、TRAP、NFATcl화CPK기인mRNA함량분별위4.980 ±0.180、3.128±0.143、6.166±0.223화4.922±0.163,여R +Ti조화PGE2조지간적차이유통계학의의(P＜0.05).결론 예선급여선택성COX-2억제제Cel능감소PGE2분비,억제RANK적표체,조단Ti유도적OC활화.
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 and Ti particles than in the absence of RANKL and Ti particles.When 100 μg/L celecoxib was added to the culture system,in combination with RANKL and Ti particles,the mRNA expression levels of RANK,TRAP,NFATc1 and CPK were significantly decreased.In addition,the suppressive effects of osteoclastogenesis by celecobix could be reversed by PGE2.Conclusion These results provide direct evidence that COX-2-dependent PGE2 induced by RANKL and Ti particles is required for osteoclastogenesis and suggests that reduced production of PGE2 by inactivation of COX-2 would provide a promising therapeutic target for the treatment of osteoclastogenesis induced by Ti particles.