摘要
目的:研究钙离子/钙调蛋白依赖性蛋白激酶II(Ca MKII)δ在破骨细胞分化不同阶段的表达规律,以揭示其在破骨细胞分化中的作用。方法:应用50μg/L核因子κB受体激活因子配体(RANKL)诱导小鼠RAW264.7细胞向破骨细胞分化;通过抗酒石酸酸性磷酸酶(TRAP)染色及骨磨片吸收陷窝检测评价破骨细胞生成情况;同时于诱导第0、1、3、5天末通过免疫荧光细胞化学、RT-q PCR和Western blot法检测Ca MKIIδ的mRNA及蛋白表达水平。结果:TRAP染色及骨吸收陷窝检测显示于诱导第5天有多核破骨细胞生成。第0、1、3、5天Ca MKIIδ的mRNA表达水平分别为1.028±0.041、2.478±0.087、10.524±1.284和42.914±2.667,蛋白相对水平分别为0.762、0.963、1.802和3.136,免疫荧光细胞化学检测显示Ca MKIIδ的荧光6强度呈时间依赖性递增。结论:Ca MKIIδ的表达随破骨细胞分化逐步增高,提示Ca MKIIδ在破骨细胞分化中可能起着关键调控作用。
AIM ： To study the expression profiles and the role of Ca^2 ＋ / calmodulin-dependent kinase II delta （CaMKIIδ） during osteoclast differentiation. METHODS： Mouse RAW264 . 7 cells were induced by receptor activator of nuclear factor κB ligand （ RANKL） at 50 μg/L for osteoclastogenesis. Tartrate-resistant acid phosphatase （TRAP） staining and bone resorption lacunae examination were performed to verify osteoclast formation. The expression of CaMKIIδ at mR- NA and protein levels was also determined by immunofluorescent cytochemistry, RT-qPCR and Western blot at days 0,1, 3 and 5. RESULTS ： TRAP positive multinuclear cells with bone resorption function were formed after 5 d of induction. The mRNA levels of CaMKIIS detected by RT-qPCR were 1. 028 ±0. 041, 2. 478 ±0. 087, 10. 524 ± 1. 284 and 42. 914 ± 2. 667 at days 0,1,3 and 5, respectively, while the protein levels of CaMKIIS detected by Western blot were 0. 762, 0. 963 , 1. 802 and 3. 136, respectively. The changes of protein level were also verified by immunofluorescence cytochemistry, in which the fluorescence intensity increased in a time-dependent manner （P 〈0.05）. CONCLUSION： The expression of CaMKIIδ increases with the differentiation of osteoclasts. CaMKIIδ may play a key role in the osteoclastogenesis.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2016年第10期1870-1874,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81270965)
