CaMKⅡγ在破骨细胞分化中的表达规律

摘要

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 osteodasts 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.%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/mL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CaMKⅡγ mRNA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,CaMKⅡγ mRNA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CaMKⅡγ mRNA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.