钙离子/钙调蛋白依赖性蛋白激酶ⅡδRNA干扰对下游基因表达及破骨细胞分化的影响

Influence of Ca MKⅡδ RNA interference on expression of its downstream molecules and osteoclast differentiation

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 determine the effect of Ca^2＋/Calmodulin dependent kinase Ⅱδ （CaMKⅡδ） RNA interference on the gene expression of downstream genes and osteoclast differentiation in order to elucidate its role in the differentiation. Methods Lentivirus plasmids of recombinant CaMKⅡδRNA interference were constructed, and the best multiplicity of infection （MOI） value and transfection efficiency was determined. Mouse RAW264.7 cells were divided into 3 groups： control group, blank vector group and interference group. In 5 d after virus transfection, the cells were harvested and interference efficiency was determined. Then the effects of CaMKⅡδ RNA interference on the expression of nuclear factor of activated T cells type c1 （NFATc1）, tartrate-resistant acid phosphotase （TRAP） and non-receptor tyrosine kinase （also known as SRC proto-oncogene, c-Src）, and on osteoclast differentiation were determined. Results CaMKⅡδ RNA interference vector was constructed successfully, with the best MOI value of 30, and transfection efficiency up to 78% （P〈0.05）. The interference efficiency was larger than 78% at mRNA level and 70% at protein level （P〈0.01）. CaMKⅡδ RNA interference significantly decreased the gene expression levels of NFATc1, TRAP and c-Src by 47.8%, 43.3% and 48.5% respectively at mRNA level （P〈0.01, P〈0.05）, and by 61.1%, 48.2% and 39.6%, respectively at protein level （P〈0.01）, when compared with the other 2 groups. Immunofluorescent cytochemistry obtained similar results. CaMKⅡδ RNA interference also obviouisly decreased osteoclast formation and bone absorption. Compared with the other 2 groups, the number of osteoclasts, and the number and size of absorbed lacunae were reduced by more than 49.8%, 47.6% and 61.3% respectively （P〈0.01） in the interference group. Conclusion CaMKⅡδ RNA interference significantly down-regulates the expression of NFATc1, TRAP and c-Src and inhibits osteoclast differentiation. CaMKⅡδ may play a key role in the process of osteoclast differentiation.