[0.5 Gy X-ray radiation promotes osteoblast differentiation by Wnt/β-Catenin signaling].

Objective: To investigate the important roles of Wnt signaling in the processes of 0.5Gy X-ray promoting osteoblast differentiation, and make clear the molecular mechanisms involved. Methods: Flow cytometry was employed to detect the apoptosis after osteoblast exposure to 0.5 Gy X-ray radiation.The protein level of osteoblast differentiation markers, such as collagen Iα (Col1α), alkaline phosphatase (ALP), osteocalcin (OCN), were detected by Western-blot and ALP activity staining was performed. Real-time PCR and Western-blot were utilized to evaluate the variations of key factors in Wnt signaling pathways, while specific inhibitor of Wnt/β-Catenin, XAV939 was used to block the Wnt signaling. Results: Low-dose (0.5 Gy) X-ray induced significant decline in MC3T3-E1 osteoblast apoptosis at three days after radiation.The dynamic variations in the expression of osteoblast differentiation markers, including Col1α, ALP, OCN, were observed after 0.5 Gy X-ray irradiation by Western blot analysis.The protein levels of Col1α have a reduction temporarily at 4 days of radiation (34.5%±5.8%, t=9.912, P<0.001), then a significant increase is detected at 10 day after radiation (162.5%±6.5%, t=2.673, P<0.05). OCN levels dropped by 83% (t=3.968, P<0.01) at 4 day after 0.5 Gy X-ray radiation, and raised at 10 day (39.5%±4.1%, t=3.219, P<0.05) and 14 day (79.4%±7.5%, t=6.708, P<0.001), respectively. ALP levels increased at 7 day (79.7%±22.3%, t=6.257, P<0.001) and 10 day(128.3%±6.1%, t=4.340, P<0.01)after radiation. At the same time, 0.5 Gy X-ray radiation can activate Wnt/GSK-3/β-Catenin signaling.The mRNA levels of Wnt3a、LPR5 and TCF-4 increased by 1.7 fold (t=6.573, P<0.001), 1.1 fold (t=5.323, P<0.05) and 1.4 fold (t=3.054, P<0.05) at 7 day after radiation.In addition, p-GSK-3β level reduced by 42.1% (t=4.460, P<0.01), and active β-Catenin increased by 1.9 fold (t=3.528, P<0.05). However, the specific inhibitor of Wnt/β-Catenin, XAV939 completely abrogated Wnt/β-Catenin signaling and the increase in ALP expression and activity induced by 0.5 Gy X-ray radiation. Conclusion: These results demonstrated that low dose X-ray radiation promoted osteoblast survival at early differentiation, and promoted differentiation at middle and late stage, in which Wnt signaling participated the regulation processes.目的： 研究Wnt/β－catenin信号通路在0.5 GyX线促进成骨细胞分化中扮演的重要作用及分子机制。 方法： 成骨细胞接受0.5 Gy线照射后流式细胞仪检测细胞凋亡，通过检测检测骨分化标志物Col1α、ALP、OCN蛋白表达水平评估成骨细胞分化情况，应用实时聚合酶链反应(real－time PCR)及免疫印迹检测Wnt信号通路中关键分子的表达，评估特异性抑制剂XAV939阻断Wnt/β－Catenin通路后0.5 Gy X线照射对成骨细胞分化的影响。 结果： 0.5 GyX线照射导致MC3T3－E1细胞骨分化标志物蛋白表达发生动态变化。0.5 GyX线照射后4 d成骨细胞Col1α蛋白表达水平下降(34.5%±5.8%, t＝9.912，P<0.001)，照射后10 d表达增加(162.5%±6.5%，t＝2.673，P<0.05)。OCN在照射后4 d下降约83%(t＝3.968，P<0.01)，10 d和14 d明显增加，分别为(39.5%±4.1%，t＝ 3.219，P<0.05)、(79.4%±7.50%, t＝ 6.708，P<0.001)。ALP的表达水平在7 d(79.7%±22.3%，t＝6.257, P<0.001)和10 d(128.3%±6.1%，t＝ 4.340, P<0.01)时明显增加。X线照射后7 d成骨细胞Wnt3a、LPR5及TCF－4的mRNA表达水平分别增加1.7倍(t＝ 6.573, P<0.001)、1.1倍(t＝ 5.323, P<0.05)和1.4倍(t＝ 3.054, P<0.05)；而磷酸化GSK－3β降低42.1%(t＝ 4.460, P<0.01)，稳定活化形式β－Catenin(ABC，脱磷酸β－Catenin)增加1.9倍(t＝ 3.528, P<0.05)。应用Wnt/β－catenin特异性抑制剂XAV939可以阻断X射线照射导致的ALP表达水平及活性增加。 结论： 低剂量X线照射通过Wnt/β－catenin信号通路增加成骨细胞骨分化标志物的表达、促进其分化成熟。.