果蝇裸露角蛋白2对大鼠牙囊细胞成骨向分化的调控机制研究

Nkd2 promotes the osteoblast differentiation of rDFCs through Wnt/β-catenin signaling pathway in vitro

目的:研究体外果蝇裸露角蛋白2(naked cuticle homolog 2,Nkd2)对大鼠牙囊细胞(rat dental follicle cells,rDFCs)成骨向分化的调控机制。方法:体外培养rDFCs,鉴定其组织来源并进行成骨向分化诱导,茜素红染色验证其成骨向分化能力,激光共聚焦检测检测Nkd2在中rDFCs的表达;采用小RNA干扰技术,干扰rDFCs中的Nkd2表达后进行成骨向分化诱导,western blot检测小RNA干扰对rDFCs表达成骨向分化因子Runx2、Col-1和OCN的影响;免疫荧光染色检测β-catenin在rDFCs中的分布变化,western blot检测小RNA干扰对rDFCs表达蓬乱蛋白1(Dishevelled-1,Dvl-1)的表达变化和Wnt/β-catenin信号通路激活剂Wnt3a与阻断剂FH535处理rDFCs后Nkd2的表达改变。结果:获得体外培养的rDFCs,并成功诱导其成骨向分化,茜素红染色显示矿化结节形成。Nkd2在rDFCs中的表达主要分布于胞浆和胞核周围。Nkd2小RNA干扰后的rDFCs中Runx2、Col-1蛋白表达明显下调,Dvl-1的表达下调。同时,Wnt3a处理组rDFCs中Nkd2蛋白表达量升高,而FH535处理组的表达量下降。结论:Nkd2通过Dvl-1调控Wnt/β-catenin信号通路促进rDFCs的成骨向分化;Nkd2是Wnt/β-catenin信号通路的靶基因,对Wnt/β-catenin信号通路起正反馈调节作用。

Objective： To study the function and the mechanism of Nkd2 in the osteogenic differentiation of rDFCs in vitro.Methods： The rDFCs were cultured in vitro, the tissue origin of the cells were identified and osteogenic differentiation was induced. The formation of mineralization nodules was detected by alizarin red staining. Confocal laser scanning microscopy was used to detect the expression of Nkd2 in rDFCs in vitro. rDFCs were transfected by small interfering RNA（si RNA）.Western blot analysis were used to explore the effect of Nkd2 on osteogenic differentiation factors, such as Runx2、Col-1 and OCN. The expression of β-catenin in rDFCs was detected by Immunofluorescence. The expression of Dvl-1 in rDFCs transfected by si RNA and the change of Nkd2 in rDFCs after treated by Wnt3 a or FH535 were tested by western blot analysis.Results： We obtained rDFCs in vitro which could be induced to osteogenic differentiation. Alizarin red staining showed mineralization nodules formed by rDFCs after osteogenic induction. Nkd2 was mainly distributed in the cytoplasm and around the nucleus of DFCs. Western blot revealed significantly reduce of Runx2、Col-1 protein in Nkd2-silenced rDFCs.Immunofluorescence reveled less expression of β-catenin in Nkd2-silenced rDFCs cell nucleus, and the level of Dvl-1 protein was down-regulated in Nkd2 si RNA transfecting rDFCs. The Nkd2 expression in rDFCs was significantly increased after treated by Wnt3 a, while decreased by FH535. Conclusion： Nkd2 could promote the osteogenic differentiation of rDFCs through Wnt/β-catenin signaling pathway by increasing the expression of Dvl-1. And Nkd2 is a target gene of Wnt/β-catenin signaling pathway. The expression of Nkd2 could be promoted by the activation of Wnt/β-catenin signaling pathway.