ERK5信号通路调控小鼠巨噬细胞破骨分化的实验研究  

Experiment on the regulation of ERK5 signaling pathway for the osteoclast differentiation of mice macrophages

作　　者：殷悦[1] 郭开今[2] 李超[1] 吴继彬[2] 张新珠[2] 

机构地区：[1]徐州医学院研究生学院2012级,江苏徐州221002 [2]徐州医学院附属医院,江苏徐州221002

出　　处：《中国矫形外科杂志》2015年第11期1020-1024,共5页Orthopedic Journal of China

基　　金：国家自然科学基金资助项目(编号:51075387)

摘　　要：[目的]探讨ERK5信号通路在RANKL诱导小鼠巨噬细胞分化为破骨细胞过程中的作用。[方法]体外构建RANKL诱导小鼠巨噬细胞分化成破骨细胞的实验模型,采用western blot检测不同时间点p-erk5表达量(0、15、30min,1、2、6 h);免疫荧光观察p-erk5蛋白向细胞核内转位情况;CCK-8确定药物组添加ERK 5抑制剂BIX02189的不同浓度;不同浓度BIX02189(0.3μmol/L、1.0μmol/L、3.0μmol/L)与RANKL诱导的RAW 264.7细胞共培养6 d,采用PCR检测TRAP的基因表达量。[结果]Western blot检测结果:加入RANKL 15 min后pERK5表达量开始增加,30 min后达到高峰(P<0.01),之后慢慢下降,且磷酸化的ERK5蛋白逐渐开始出现向细胞核内转位;CCK-8检测细胞增殖率结果显示,浓度在3.0μmol/L以下的BIX02189对小鼠巨噬细胞正常增殖不构成影响;不同浓度的BIX02189对破骨细胞特异性基因TRAP表达量,较对照组有较明显抑制作用,且TRAP的基因表达随着浓度的增加有不同程度的减少(P<0.05)。[结论]RANKL能够激活RAW264.7细胞的ERK5信号通路,且抑制ERK5通路后,能够一定程度减少巨噬细胞向破骨细胞分化。[ Objective] To explore the role of ERK5 pathway in the process RANKL induces mice macrophage to differen- tiate into osteoclasts. [ Methods] An experimental model in which RANKL induces mice macrophages to differentiate into osteo- clasts was built in vitro. Western blot was adopted to test the expression quantity of p- erk5 at different time points （0 min, 15 miu, 30 min, 60 min, 2 h, 6 h） . The translocation of p - erk5 protein into the nucleus was observed by immunofluores- cence. Different concentrations of ERK5 inhibitors BIX02189were determined by CCK -8. ERK5 inhibitor BIX02189 with differ- ent concentrations （0. 3 μmol/L, 1.0 μmol/L, 3.0 μmol/L） was added with RANKL - induced RAW264. 7 cells were co - cultured for 6 days. PCR was adopted to test the genetic expression quantity. [ Results] 15 min after RANKL was added, the p - ERK5 expression increased and reached a peak value in 30 rain. After 2 h, the expression quantity decreased and phosphoryl- ated ERK5 protein began to transfer into the nucleus. CCK - 8 detection cell proliferation rate, the results indicated that concen- tration under 3.0 u mol/L BIX02189 have no effects on mouse macrophage normol proliferation. BIX02189 with different concen- trations has obvious inhibitory effect on the osteoclast control group. Besides, the genetic expression of TRAP decreased to vari- ous degrees with the increase of concentration. [ Conclusion ] RANKL can activate the ERK5 pathway in RAW264.7 cells, and after inhibiting ERK5 pathway, it can reduce the osteoclast differentiation of macrophages to a certain degree.

关 键 词：ERK5 核因子-ΚB受体活化因子配体 小鼠巨噬细胞 BIX02189 

分 类 号：R392[医药卫生—免疫学]