CAJ | 학술논문

目的观察Ⅱ型大麻素受体(CB2)选择性抑制剂AM630对钛颗粒(Ti)诱导炎性破骨细胞(OC)活化的影响.方法实验分5组,即空白组、核因子-κB (NF-κB)受体活化因子配体(RANKL)组、Ti组、R+Ti组、药物组.采用噻唑蓝(MTT)法检测0.1 g/LTi颗粒和不同浓度AM630(50、100、200 nmol/L)处理小鼠单核/巨噬细胞株RAW264.7后24、48、72 h细胞增殖活性；以0.1 g/L Ti颗粒和(或)50 μg/L RANKL诱导RAW264.7,6d时加入AM630再培养24h,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CB2、NF-κB受体活化因子(RANK)、肌酸磷酸激酶(CPK)基因mRNA含量,酶联免疫吸附试验(ELISA)检测炎性因子白细胞介素(IL)-1β和肿瘤坏死因子(TNF)-α表达水平.结果 MTT结果表明0.1 g/L Ti颗粒和(或)AM630(50、100、200 nmol/L)对RAW264.7细胞增殖能力无影响.TRAP染色,RANKL组、R+Ti颗粒组均可见大量的紫红色多核细胞；Ti组、药物组阳性细胞较少,统计分析结果表明AM630≥100 nmol/L时,TRAP阳性细胞数量与RANKL组[(181.80±14.23)/孔]比较差异有统计学意义(p＜0.05).定量RT-PCR结果显示R+Ti组CB2、RANK以及CPK mRNA含量分别为11.26±1.39、9.68±0.91和9.93±0.56；药物组(100 nmol/L AM630)上述基因mRNA含量为4.73±0.55、3.85±0.45和5.42±0.87,差异有统计学意义(P＜0.05).ELISA结果显示,Ti颗粒加入24h后IL-1β、TNF-α的含量分别为(176.9±9.2)、(159.7±8.2) ng/L;与药物组[IL-1β:(105.5±7.0) ng/L,TNF-α:(75.9±8.1) ng/L]比较,差异有统计学意义(P＜0.05).结论 Ⅱ型大麻素受体选择性抑制剂AM630能够抑制Ti颗粒引起的炎性破骨细胞活化.
목적 관찰Ⅱ형대마소수체(CB2)선택성억제제AM630대태과립(Ti)유도염성파골세포(OC)활화적영향.방법 실험분5조,즉공백조、핵인자-κB (NF-κB)수체활화인자배체(RANKL)조、Ti조、R+Ti조、약물조.채용새서람(MTT)법검측0.1 g/LTi과립화불동농도AM630(50、100、200 nmol/L)처리소서단핵/거서세포주RAW264.7후24、48、72 h세포증식활성；이0.1 g/L Ti과립화(혹)50 μg/L RANKL유도RAW264.7,6d시가입AM630재배양24h,이항주석산산성린산매(TRAP)염색검측성숙OC,이역전록-취합매련반응(RT-PCR)검측CB2、NF-κB수체활화인자(RANK)、기산린산격매(CPK)기인mRNA함량,매련면역흡부시험(ELISA)검측염성인자백세포개소(IL)-1β화종류배사인자(TNF)-α표체수평.결과 MTT결과표명0.1 g/L Ti과립화(혹)AM630(50、100、200 nmol/L)대RAW264.7세포증식능력무영향.TRAP염색,RANKL조、R+Ti과립조균가견대량적자홍색다핵세포；Ti조、약물조양성세포교소,통계분석결과표명AM630≥100 nmol/L시,TRAP양성세포수량여RANKL조[(181.80±14.23)/공]비교차이유통계학의의(p＜0.05).정량RT-PCR결과현시R+Ti조CB2、RANK이급CPK mRNA함량분별위11.26±1.39、9.68±0.91화9.93±0.56；약물조(100 nmol/L AM630)상술기인mRNA함량위4.73±0.55、3.85±0.45화5.42±0.87,차이유통계학의의(P＜0.05).ELISA결과현시,Ti과립가입24h후IL-1β、TNF-α적함량분별위(176.9±9.2)、(159.7±8.2) ng/L;여약물조[IL-1β:(105.5±7.0) ng/L,TNF-α:(75.9±8.1) ng/L]비교,차이유통계학의의(P＜0.05).결론 Ⅱ형대마소수체선택성억제제AM630능구억제Ti과립인기적염성파골세포활화.
Objective To explore the effect of cannabinoid receptor 2 (CB2) selective antagonist (AM630) 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 AM630 and Ti particles on RAW264.7 cell viability was examined by using the methyl thiazol tetrazolium (MTT) assay.Osteoclast formation was measured by tartrate resistant acid phosphatase (TRAP) staining using a commercial kit.The mRNA levels of CB2,creatine phosphokinase(CPK) and NF-κB receptor activation factor(RANK) were detected by using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR).Enzyme-linked immunosorbent assay (ELISA)was performed to determine interleukin (IL)-1β and tumor necrosis factor (TNF)-t.Results AM630 (0-200 nmol/L) did not affect viability of RAW264.7 cells cultured with Ti particles.The mature osteoclasts were obtained from RAW264.7 cells stimulated with RANKL and Ti particles for 6 days and detected by TRAP staining.AM630 at a doses of ≥ 100 nmol/L significantly reduced the number of TRAP-positive cells as compared with controls.After culture for 6 days,mRNA levels of CB2,RANK and CPK were significantly higher in the media with RANKL and Ti particles than in those without RANKL and Ti particles.When 100 nmol/L AM630 in combination with RANKL and Ti particles was added to the culture system,mRNA levels of CB2,RANK and CPK were significantly decreased.The concentrations of IL-1β and TNF-α in RAW264.7 cell cultures were measured by using ELISA.After culture for 24 h,IL-13 and TNF-α concentrations were significantly increased in the media with Ti particles as compared with those in the media withou Ti particles.The cytokine concentration was further increased with culture time.Conclusion AM630 could significantly inhibit Ti particle-induced inflammatory osteoclastogenesis.