CaMKⅡγ在破骨细胞分化中的表达规律

Expression profiles of CaMKⅡγ during osteoclast differentiation

目的:研究钙离子/钙调蛋白依赖性激酶γ(CaMKⅡγ)在破骨细胞分化中的表达规律,以揭示其作用。方法:用50 ng/m L核因子-κB受体活化因子配体(RANKL)诱导RAW264.7细胞向破骨细胞分化,于诱导后第0,1,3,5天收获细胞,用抗酒石酸酸性磷酸酶(TRAP)染色评价破骨细胞生成情况,并用RT-PCR、Western印迹、免疫荧光细胞化学法检测CaMKⅡγm RNA和蛋白表达。结果:多核破骨细胞在诱导后第3天开始出现,第5天达到最多。分化第0,1,3,5天,CaMKⅡγm RNA相对表达水平分别为(1.067±0.179),(1.840±0.070),(9.493±0.453)和(30.767±0.573);蛋白相对表达水平分别为(0.454±0.065),(0.613±0.021),(0.858±0.019)和(0.980±0.023);除第1天的蛋白相对水平外,其他时间点CaMKⅡγm RNA和蛋白表达差异均有统计学意义,且呈时间依赖性增强(P<0.01)。免疫荧光细胞化学法检测也显示CaMKⅡγ蛋白表达在破骨细胞分化过程中逐步增加。结论:CaMKⅡγ表达随破骨细胞分化呈时间依赖性增加,提示其在破骨细胞分化中可能发挥关键调控作用。

Objective： To study the expression profiles and the role of Ca2＋/calmodulin-dependent protein kinase IIγ （CaMKIIγ） during osteoclast differentiation. Methods： Mouse RAW264.7 cells were induced for osteoclastogenesis with 50 ng/mL receptor activator of nuclear factor-κB ligand （RANKL） and the cells were harvested at 0, 1, 3 and 5 days after induction. Tartrate-resistant acid phosphotase staining was performed to verify osteoclasts formation. RT-PCR, Western blot and immunofluorescent cytochemistry were used to detect the CaMKIIγ gene expression during osteoclastogenesis.Results： The osteoclasts were formed at day 3 under RANKL induction and more osteoclasts were observed at day 5. At day 0, 1, 3 and 5, the relative level of CaMKIIγ mRNA were （1.067±0.179）,（1.840±0.070）, （9.493±0.453） and （30.767±0.573）, respectively, and the relative protein level were （0.454±0.065）, （0.613±0.021）, （0.858±0.019） and （0.980±0.023）, respectively. CaMKIIγ expression was increased in a time-dependent manner except relative protein level at day 1 （P〈0.01）,which showed no significant difference at day 0 （P〉0.05）. Immunofluorescence assay showed that CaMKIIγ protein was also increased with differentiation of osteoclasts. Conclusion： The CaMKIIγ expression was increased in a time-depended manner during osteoclast differentiation and it might play a vital role during osteoclastogenesis.