大麻素受体CB2选择性抑制剂对RAW264．7细胞分化为破骨细胞的作用  

The role of eannabinoid receptor 2 selective antagonist in osteoclast differentiation of RAW264. 7 cells

作　　者：耿德春[1] 徐耀增[1] 朱雪松[1] 王骏骅[1] 王根林[1] 杨惠林[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》2011年第5期784-786,共3页Chinese Journal of Experimental Surgery

基　　金：江苏省高校自然科学基金资助项目（Q2122707）;江苏省卫生厅科研资助项目（H201012）;苏州市社会发展计划资助项目（SS0809）

摘　　要：目的观察大麻素受体CB2选择性抑制剂-AM630对核因子（NF）-κB受体活化因子配体（RANKL）诱导的小鼠单核／巨噬细胞株RAW264．7向破骨细胞分化的影响。方法实验分3组，即空白组，诱导组和药物组。采用噻唑蓝（MaT）法检测不同浓度AM630（0、50、103、200nmol／L）刺激RAW264．7后24、48、72h的细胞增殖活性。以50μg/L RANKL诱导RAW264．7，6d后加入103nmoL／LAM630再培养24h；抗酒石酸酸性磷酸酶（TRAP）染色检测成熟破骨细胞，定量逆转录-聚合酶链反应（RT—PCR）测量CPK、RANK基因mRNA含量，Westernblot检测ERK及p-ERK表达水平。结果M1rr结果表明AM630浓度为50、103、200nmol／L时对RAW264．7细胞增殖能力无影响。TRAP染色结果表明药物组成熟破骨细胞数（65．60±4．83）／em2明显少于诱导组（181．00±6．86）／cm2，差异有统计学意义（P〈0．05）。定量RT—PCR结果证实，诱导组CPK和RANK基因mRNA含量分别为18．50±5．12和12．70±2．61；加入AM630后，上述基因mRNA含量为7．00±1．03和4．80±1．25；差异有统计学意义（P〈0．05）。Westernblot检测显示，AM630能下调RANKL诱导的p-ERK表达水平。结论大麻素受体CB2选择性抑制剂-AM630能有效地抑制RANKL诱导的RAW264．7向破骨细胞分化。Objective To observe the effect of cannabinoid receptor 2 selective antagonist-AM630 on receptor activator of NF-KB ligand （RANKL）-induced osteoclast differentiatioin using the monocytemacrophage cell line RAW264. 7. Methods The experiment involved 3 groups ： black group, induced group and treatment group. Methylthiazol tetrazolium （MTT） assay was used to analyze the viability of RAW264.7 cells which were exposed to different concentrations of AM630 （0, 50, 100, 200 nmol/L）. RAW264. 7 cells were plated at a density of 104 cells/well in slx-well tissue culture plate and incubated with or without RANKL for 6 days, then 100 nmol/L AM630 was added for another 24 h. Osteoelast formation was measured by tartrate resistant acid phosphatase （TRAP） staining using a commercial kit. The mRNA expression levels of CPK and RANK were measured by using quantitative real-time reverse transerlption-polymerase chain reaction （RT-PCR）. The extracellular regulated kinase （ERK） pathway was determined by Western blotting. Results AM630 （50-200 nmol/L） did not affect RAW264. 7 cell viability. Mature osteoclasts were obtained from RAW264. 7 cells stimulated with RANKL for 6 days and detected by TRAP staining. AM630 could significantly inhibit the formation of TRAP-positive cells （ P 〈 0. 05 ）. The mRNA expression levels of CPK and RANK were significantly increased when the cells were treated with RANKL for 6 days. AM630 treatment resulted in an obvious reduction of CPK and RANK gene transcripts as compared with untreated group （ P 〈 0. 05 ）. Treatment with RANKL markedly induced the phosphorylation of ERK. The increase in ERK phosphorylation was inhibited by the treatment of cells with AM630. Conclusion Cannabinoid receptor 2 selective antagonist could inhibit the osteoclast differentiation of mature osteoclasts from RAW264. 7 induced with RANKL.

关 键 词：大麻素受体 破骨细胞 RANKL 

分 类 号：R965[医药卫生—药理学]