CaMKⅡδ慢病毒过表达载体构建及其对破骨细胞分化的影响

Construction of the recombinant CaMKⅡδ lentivirus vector and its effect on differentiation of osteoclast

目的研究钙离子/钙调蛋白依赖性激酶Ⅱδ(CaMKⅡδ)过表达对破骨细胞分化的影响。方法筛选破骨细胞分化中CaMKⅡδ高表达转录本,构建过表达载体。RAW264.7细胞分成对照组、空载体组和过表达组,检测CaMKⅡδ表达情况;并在分化诱导5 d后检测破骨细胞生成及骨吸收情况。结果 CaMKⅡδ转录本2和3在破骨细胞分化中高表达;用转录本2构建了过表达载体,建立了稳定转染株。过表达组CaMKⅡδ mRNA水平较对照组、空载体组分别上升107.8%和85.7%(P<0.05),蛋白水平分别上升37.2%和37.1%(P<0.05),证实重组CaMKⅡδ在细胞中得到有效表达。CaMKⅡδ过表达组破骨细胞数目、牙本质吸收陷窝数和面积与对照组和空载体组比较差异均无统计学意义(P>0.05)。结论 CaMKⅡδ过表达对破骨细胞分化和骨吸收无明显影响。

Objective To investigate the effect of Ca2+/Calmodulin dependent kinase IIδ(CaMKIIδ)over-expression on differentiation of osteoclast.Methods CaMKIIδtranscript variant was highly expressed during osteo-clast differentiation,and was amplified and used for construction of recombinant lentivirus vector.RAW264.7 cells stably expressing the recombinant CaMKIIδwere established.The cells were divided into the control group,the blank vector group and CaMKIIδoverexpression group.CaMKIIδexpression was detected in RAW264.7 cells.After 5 day osteoclastgenic induction,osteoclastogenesis and bone resorption function were detected.Results CaMKIIδtranscript variant 2 and 3 were highly expressed during osteoclast differentiation and the recombinant CaMKIIδtran-script variant 2 overexpression vector was constructed.Compared to the control and blank vector,mRNA level of CaMKIIδin the overexpression group was increased about 107.8%and 85.7%(P<0.05,respectively).Protein level of CaMKIIδwas increased about 37.2%and 37.1%(P<0.05,respectively).After osteoclastgenic induc-tion,no significant differences in number of osteoclasts,number and size of dentin absorption lacunaes were observed between the control group and the blank vector group.Conclusion CaMKIIδexhibited no significant effect on osteoclast differentiation.