ABSTRACT
Objective To investigate the effects of calmodulin-dependent kinase IIγ (GaMKIIγ) RNA interference on osteoclast differentiation and bone resorption. Methods Three CaMKIIγ recomninant RNA interference vectors were constructed using lentivirus. Negative vector was used to transfect RAW264.7 cells and the multiplicity of infection (MOI) with the optimal transfection efficiency was determined. Recombinant vectors were also used to transfect cells to determine the one with the best interference effect for following experiments.Then, the cells were divided into control group, negative vector group and interference vector group. Five days after virus transfection, osteoclastogenesis and bone resorption function were determined by TRAP staining and dentin resorption lacunae detection. Results Three CaMKIIγ recombinant interference vectors were constructed, and the optimal MOI was 30, under which transfection efficiency was about 81％. The #3 recombinant vector showed the best interference effect and the interference efficiency was up to 78.16％ at mRNA level and 67.02％ at protein level. When compared with control group, the number of multi-nucleated osteoclasts, the number and area of dentin resorption lacunaes in interference vector group decreased 59.99％、54.19％ and 57.94％ respectively (P < 0.01). No significant difference were observed between negative vector group and control group (P> 0.05).Conclusion CaMKIIγ RNA interference significantly inhibits osteoclastogenesis and bone resorption.
ABSTRACT
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.