Notch1蛋白高表达抑制破骨细胞增殖和分化

Up-regulation of Notch1 inhibits proliferation and differentiation of osteoclast in vitro

目的探究体外培养条件下Notch1蛋白高表达对细胞核因子κB受体活化因子配体(RANKL)和巨噬细胞集落刺激因子(MCSF)诱导的骨髓源破骨细胞增殖分化的影响。方法体外培养Rosa-notch1转基因小鼠骨髓间充质干细胞(BMSCs),采用RANKL和MCSF刺激BMSCs向破骨细胞方向分化。培养至第3天,实验组和对照组分别转染携带Cre重组酶和绿色荧光蛋白(GFP)的重组腺病毒(Ad-Cre-GFP)和携带GFP的重组腺病毒(Ad-GFP),启动实验组Notch1高表达,采用实时聚合酶链反应(RT-PCR)检测Notch信号通路成分(Notch1、Notch2、Notch3、Notch4、Delta1、Delta3、Delta4、Jagged1)、靶基因(Hes1)m RNA表达量以及抗酒石酸酸性磷酸酶(TRAP)在诱导后m RNA的表达量。采用TRAP染色法观察破骨细胞增殖分化情况。结果 TRAP染色显示实验组破骨细胞形成数量明显少于对照组(P<0.05)。与对照组相比,实验组Notch1、Notch3、Jagged1、Delta3、Hes1 m RNA表达量明显增高(P<0.05),TRAP的m RNA表达量明显降低(P<0.05)。结论 Notch1高表达抑制RANKL和MCSF诱导的破骨细胞增殖分化。

Objective This study aimed to explore the effect of the up-regulation of Notch1 on osteoclastogenesis induced to osteoclasts by receptor activator for nuclear factor-κB ligand（RANKL） and macrophage colony-stimulating factors（MCSF） in vitro. Methods The bone marrow stem cells（BMSCs） of Rosa-notch1 mice were cultured and induced to osteoclasts by RANKL and MCSF. The BMSCs were transfected with the Ad-Cre-green fluorescent protein（GFP） virus or Ad-GFP virus. Total RNA from cells was extracted, and the gene expression levels of Notch1, Notch2, Notch3, Notch4, Delta1, Delta3, Delta4, Jagged1, Hes1, and tartrate resistant acid phosphatase（TRAP） were detected at the defined stage by reverse transcriptionpolymerase chain reaction（RT-PCR）. Osteoclast formation was analyzed by TRAP assay. Results The number of TRAPpositive multinuclear cells of the experimental group significantly decreased compared with that of the control group. The m RNA expression levels of Notch1, Notch3, Jagged1, Delta3, and Hes1 of the experimental group were significantly higher than those of the control group, whereas the TRAP m RNA expression of the experimental group was significantly lower than that of the control group（P〈0.05）. Conclusion Up-regulation of Notch1 inhibit osteoclastogenesis of BMSCs induced by RANKL and MCSF in vitro.