目的 研究活化T细胞核因子蛋白c1(NFATc1)过表达对唑来膦酸诱发的破骨细胞生成抑制的挽救效应.方法 小鼠NFATc1重组慢病毒转染RAW264.7细胞,验证NFATc1蛋白表达.细胞分为A、B两组,分别转染空白载体和NFATc1重组载体.两组细胞均用50 ng/mL核因子κB受体激活蛋白配体(RANKL)诱导5 d,并在第2天加入1×10-6 mmol/mL 唑来膦酸作用2 d.第6天收获细胞,检测破骨细胞的生成和功能及NFATc1、抗酒石酸酸性磷酸酶(TRAP)、酪氨酸激酶(c-Src)基因的表达情况.结果 NFATc1蛋白在细胞中均有表达.B组TRAP阳性破骨细胞数、牙本质吸收陷窝数目和面积显著高于A组(P<0.01).B组NFATc1、TRAP、c-Src的蛋白水平显著高于A组(P<0.01);B组3个基因mRNA水平显著高于A组(P<0.01).免疫荧光细胞化学检测也得到相似的结果.结论 NFATc1过表达对唑来膦酸诱发的破骨细胞生成及NFATc1、TRAP、c-Src基因表达的抑制具有挽救效应;NFATc1作为Ca2+信号的关键分子,在唑来膦酸诱发的破骨细胞生成抑制中起着关键作用.%Objective To investigate the rescue effect of nuclear factor of activated T cells type c1(NFATc1) over-expression on zoledronate-induced inhibition of osteoclastogenesis.Methods RAW264.7 cells were transfected with mouse recombinant NFATc1 lentivirus, and protein expression of NFATc1 was verified.The cells were divided into Group A and B, in which the cells were transfected with blank vector and recombinant NFATc1 vector, respectively.The cells in both groups were treated with 50 ng/mL RANKL for 5 days and 1×10-6 mmol/mL zoledronate for 48 h from Day 2.The cells were harvested on Day 6 and osteoclastogenesis, as well as gene expression of NFATc1, TRAP and c-Src were examined.Results NFATc1 protein was successfully expressed in RAW264.7 cells.TRAP positive multinuclear osteoclast count, and the number and size of absorption lacunae on dentin slices in Group B were all significantly higher than those in Group A (P<0.01).Protein levels of NFTAc1, TRAP and c-Src in Group B were also significantly higher than those in Group A (P<0.01).And mRNA levels of above three genes in Group B were significantly higher than those in Group A (P<0.01).Similar results were also achieved in immunofluorescent cytochemistry examination.Conclusion Over-expression of NFATc1 can rescue zoledronate-induced inhibition of osteoclastogenesis and up-regulate gene expression of NFATc1, TRAP and c-Src.As a key transducer of Ca2+ signal, NFATc1 may play a key role in zoledronate-induced inhibition of osteoclastogenesis.
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