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目的研究钙离子/钙调蛋白依赖性蛋白激酶Ⅱδ(Ca2+/Calmodulin dependent kinaseⅡδ,Ca MKⅡδ)RNA干扰对下游基因表达及破骨细胞分化的影响,以证实其在破骨细胞分化中的作用。方法应用慢病毒构建Ca MKⅡδRNA干扰载体,并确定最佳感染复数(multiplicity of infection,MOI)值及转染效率。小鼠RAW264.7细胞分为对照组、空白载体组和干扰组。转染5 d后收获细胞,确定干扰效率;并检测RNA干扰对活化T细胞核因子蛋白c1(NFATc1)、抗酒石酸酸性磷酸酶(TRAP)、非受体酪氨酸激酶(c-Src)基因表达及破骨细胞分化的影响。结果成功构建了Ca MKⅡδRNA干扰载体,最佳转染MOI值为30,转染效率可达78%(P<0.05)。重组病毒对Ca MKⅡδ的干扰效率在mRNA水平超过78%,在蛋白水平超过70%(P<0.01)。Ca MKⅡδRNA干扰显著降低NFATc1、TRAP和c-Src基因,与对照组、空白载体组比较,其mRNA水平下降分别超过了47.8%、43.3%和48.5%(P<0.05,P<0.01),蛋白相对水平分别超过了61.1%、48.2%和39.6%%(P<0.01);免疫荧光细胞化学检测也得到相似结果。Ca MKⅡδRNA干扰也显著降低了破骨细胞生成及骨吸收功能;与其他两组比较,干扰组破骨细胞数、牙本质吸收陷窝数目和面积下降分别超过了49.8%、47.6%和61.3%(P<0.01)。结论 Ca MKⅡδRNA干扰可显著下调其下游NFATc1、TRAP、c-Src基因表达并抑制破骨细胞分化;Ca MKⅡδ在破骨细胞分化中可能发挥关键调控作用。
Objective To investigate the effects of Ca 2+ / Calmodulin-dependent kinase Ⅱδ (Ca MKⅡδ) RNA interference on downstream gene expression and osteoclast differentiation in order to confirm its role in osteoclast differentiation. Methods The lentivirus was used to construct Ca MK Ⅱ δ RNA interference vector and the optimal multiplicity of infection (MOI) value and transfection efficiency were determined. Mouse RAW264.7 cells were divided into control group, blank vector group and interference group. After 5 days of transfection, the cells were harvested and the interference efficiency was determined. The effects of RNA interference on the expression of NFATc1, TRAP and c-Src in activated T cells And osteoclast differentiation. Results Ca MK Ⅱ δ RNA interference vector was successfully constructed. The best transfection MOI was 30 and the transfection efficiency was 78% (P <0.05). The interference efficiency of recombinant virus on CaMKIIδ was above 78% at mRNA level and over 70% at protein level (P <0.01). Ca MKⅡδRNA significantly decreased the expression of NFATc1, TRAP and c-Src genes compared with the control and blank vector groups (47.8%, 43.3% and 48.5% respectively) (P <0.05, P <0.01) Levels exceeded 61.1%, 48.2% and 39.6 %% respectively (P <0.01), and similar results were obtained by immunofluorescence cytochemistry. Compared with the other two groups, the numbers of osteoclasts and dentin absorption lacuna in the interference group decreased by 49.8%, 47.6% and 61.3% respectively (P <0.05) P <0.01). CONCLUSION: Ca MKⅡδRNA interference may significantly down-regulate the expression of NFATc1, TRAP and c-Src genes and inhibit osteoclast differentiation. Ca MKⅡδ may play a key regulatory role in osteoclast differentiation.