通过RAW264.7细胞系初步研究破骨细胞中Notch1对RANKL-RANK系统的影响

A preliminary study on the impact of Notchl on RANKL/RANK system in osteoclast via RAW264.7 cells

目的·探索细胞核因子κB受体激活蛋白配体(receptor activator of nuclear factor-κB ligand,RANKL)/核因子κB受体激活蛋白(receptor activator of nuclear factor-κB,RANK)系统及Notch通路间的交互调控机制。方法·通过RANKL (35 ng/L)刺激RAW264.7小鼠破骨细胞前体细胞系,用real-time PCR、Western blotting检测Notch通路关键分子的表达水平。小干扰RNA(small interfering RNA,si RNA)技术抑制Notch通路中Notch1、Dll1和Dll3分子的表达,检测上述分子抑制后Rank mRNA和蛋白表达的变化。结果·RANKL刺激引起Notch1、Jagged1、Jagged2的表达降低,Dll4的表达升高。RANKL抑制Notch通路下游分子Hes1、Hey1的表达。通过si RNA技术,Notch1、Dll1和Dll3 mRNA的表达分别下调71%(P=0.000)、53%(P=0.015)和70%(P=0.000)。Notch1表达抑制后,Rank m RNA和蛋白的表达均增加(P=0.003,P=0.000)。结论·Notch1对RANKL-RANK系统的潜在调节机制,可能在破骨细胞相关的溶骨活动中发挥重要作用。

Objective · To investigate the crosstalk between receptor activator of nuclear factor-KB ligand(RANKL)/receptor activator of nuclear factor-κB(RANK) system and Notch signaling pathways. Methods · Mouse osteoclast precursor cell line RAW264.7 was cultured and stimulated by RANKL(35 ng/L). Real-time PCR and Western blotting analysis were used to determine the expressions of Notch signaling molecules in RANKL-stimulated RAW264.7 cells. Small interfering RNA(siRNA) transfection was used to inhibit the expression of Notch 1, Dll1 and D113 in Notch signaling pathways,then the mRNA and protein expressions of Rank were detected using real-time PCR and Western blotting analysis, respectively. Results · The expressions of Notch1, Jagged1 and Jagged2 were down-regulated while the expression of Dll4 was up-regulated after RANKL stimulation. The stimulation also inhibited the expression of Hes1 and Hey1. After siRNA transfection, the mRNA expressions of Notch1, Dll1 and Dll3 were suppressed by 71%(P=0.000),53%(P=0.015) and 70%(P=0.000), respectively. The mRNA and protein expressions of Rank were up-regulated after Notch1 inhibition(P=0.003,P=0.000). Conclusion · The data infers that the impact of Notch1 on RANKL/RANK system might play a key role in bone resorption conducted by osteoclast.