神经肽Y对小鼠成骨细胞系细胞成骨分化的影响及其与Wnt通路相关机制的初步研究

Neuropeptide Y stimulates osteoblastic differentiation of murine MC3T3-E1 cells related to activated Wnt signaling in vitro

目的探讨神经肽Y（NPY）对小鼠成骨细胞系细胞（MC3T3．E1细胞）成骨分化的生物学效应，并从Wnt信号通路角度探讨其可能的机制。方法体外培养MC3T3．E1细胞，将不同浓度的NPY（10^-4 mol／L、10^-10mol／L、10^-12mol／L）作用于细胞，在培养的不同时期（1、3、5、7、9d）使用噻唑蓝法检测细胞增殖情况。成骨诱导条件下将不同浓度的NPY作用于MC3T3．E1细胞，在传代培养的不同时期（4、7、14d）采用免疫细胞化学、Westernblot对MC3T3．T1细胞诱导进行鉴定并确定促成骨效应最适的浓度；成骨诱导4d时将最适浓度NPY、NPY加经典wnt通路抑制因子（DKK1）、NPY加Y1受体拮抗剂分别作用于细胞，采用Westernblot法检测经典Wnt通路相关蛋白（β-cmenin和p-GSK-3β）的表达，免疫荧光检测各组β-catenin蛋白核内转移的情况。结果与对照组相比，不同浓度的NPY在7、9d时均显著促进MC3T3-E1细胞增殖。成骨诱导条件下，NPY（10^-8mol／L）和NPY（10^-10mol／L）浓度组、NPY（10^-10mol／L）和NPY（10^-12mol／L）浓度组分别在4d、14d时显著促进碱性磷酸酶（ALP）蛋白表达。NPY促成骨效应的最适浓度为10^-10mol／L。在成骨诱导4d时，使用DKKl或Yl受体拮抗剂可以显著抑制NPY（10^-10tool／L）组对ALP蛋白表达的促进作用，虽然β-cmenin和p-GSK-3β蛋白表达未受影响，但是NPY可明显激活β-cmenin的核内转移。结论NPY促进MC3T3-E1细胞成骨分化过程中ALP的蛋白表达，该效应可能是通过经典wnt信号通路介导。

Objective To investigate the effect of neuropeptide Y （NPY） on the osteoblastic dif- ferentiation of routine MC3T3-E1 cells and its mechanism related to the Wnt signaling pathway. Methods The routine MC3T3-E1 cells were divided into 4 groups according to the stimulators added： phosphate buffered saline （PBS） （control） and different concentrations of NPY （10^-8 mol/L, 10^-10 mol/L and 10^-12 mol/L）. The cellular proliferation was detected with MTT assay after 1, 3, 5, 7 and 9 days. The cells were identified with cell immunochemistry and Western Blot to find out the most effective concentration of NPY at different time points under osteoblastic condition. The cells were then divided into 4 groups： PBS, NPY, NPY ＋ NPY receptor antagonist, and NPY ＋ DKK1. Western blot was used to determine the expression of β-catenin and p-GSK-3β in each group. Nuclear signaling activity of β-catenin was observed using immunofluorescence staining. Results NPY significantly improved the proliferation of MC3T3-E1 cells at 7 and 9 days （ P 〈 0. 05）. NPY （10^-8 mol/L and 10^-10 mol/L） groups and NPT （10^-10 mol/L and 10^-12 mol/L） groups signif- icantly improved the ALP activity at 4 and 14 clays respectively （ P 〈 0. 05） . At 4 days, the expression of ALP protein was significantly decreased in the NPY ＋ DKK1 group and the NPY ＋ NPY receptor antagonist group compared with that in the NPY group （ P 〈 0.05） . Although the expression levels of β-catenin and p-GSK-3β protein were uninfluenced in either case, NPY significantly stimulated the nuclear signaling activity of β-catenin. Conclusions NPY may significantly increase the expression of ALP protein in MC3T3-E1 cells during osteoblastic differentiation. This effect might be mediated through the canonical Wnt signaling pathway.