转化生长因子-β1／Smad2／3信号通路在O．5GyX线促进成骨细胞分化过程的作用

Transforming growth factor - β1d/Smad2/3 signaling pathway plays important roles in osteoblasts differentiation promoted by 0. 5 Gy X - ray radiation

目的观察转化生长因子-β1（TGF-β1）／Smads通路在0．5Gyx线照射促进成骨细胞分化过程的作用。方法0．5GyX线照射成骨细胞后，评估I型胶原仅（Colld）、碱性磷酸酶（ALP）、骨钙素（OCN）、Runt相关转录因子2（Runx2）、成骨细胞特异转录因子（Osterix）蛋白表达水平以及矿化功能。应用实时定量聚合酶链反应及Westernblot检测TGF-β1／Smads信号通路中关键分子TGF-β1及受体激活素受体样激酶5（ALK5）、Smad2／3的表达变化。结果0．5Gyx线照射促进MC3T3-E1成骨细胞矿化成熟。Westernblot检测显示MC3T3．E1细胞在0．5Gyx线照射后Colla、ALP、OCN、Runx2、Osterix蛋白表达水平发生动态变化。照射后4d成骨细胞TGF-β1mRNA表达水平升高1．7倍（t=8．414，P=0．000），受体ALK5mRNA在4d和7d时分别升高2．2倍（t=12．660，P=0．000）和2．5倍（t=10．380，P=0．000）。酶联免疫吸附测定（ELISA）显示x线照射成骨细胞后4d和7d培养上清中活化TGF-β1分别为（310．42±36．39）pg／ml（P=0．007）、（420．13±40．61）pg／ml（P=0．000）。在0．5Gyx线照射后7d时，成骨细胞p-Smad2的蛋白表达明显升高（80．56±7．14，P=0．009）。结论0．5Gyx线照射促进成骨细胞分化的过程中TGF-β1／Smad2／3信号通路具有重要调控作用。

Objective To investigate the roles of transforming growth factor-β1 （TGF-β1 ）/ Smads signaling pathway in osteoblasts differentiation induced by 0. 5 Gy X - ray radiation. Methods The protein levels of osteoblast differentiation markers were detected and matrix mineralization was assessed by Alizarin red staining. Real - time polymerase chain reaction and Western blotting were utilized to evaluate the variations of key factors in TGF - β1/Smads signaling pathways, including TGF-β1, activin receptor - like kinase 5 （ ALK5 ） and Smad2/3. Results The accelerated matrix mineralization was detec- ted after radiation exposure. The protein levels of type I collagen alpha （Colltα） , alkaline phosphatase （ ALP）, osteocalcin （ OCN）, Runt - related transcription factor 2 （ Runx2 ）, Osterix presented dynamic changes after 0. 5 Gy X - ray radiation. TGF - [31 mRNA levels increased by 1.7 fold at 4 d after radiation （t = 8. 414, P = 0. 000）. The mRNA expression levels of ALK5 were increased by 2. 2 fold （t = 12. 660, P = 0. 000 ） and 2. 5 fold （ t = 10. 380, P = 0. 000 at 4 d and 7 d after radiation respectively. Enzyme linked immunosorbent assay （ELISA） test show activated TGF - β1 in supernataut was （ 310. 42 ± 36. 39） pg/ml （P =0. 007） and （420. 13 ±40. 61） pg/ml （P =0. 000） at 4 d and 7 d after radiation re- spectively. The increased TGF - β1 activity has a significant difference in radiation group. In addition, p - Smad2 protein level was increased at 7 d after 0. 5 Gy X - ray radiation （ 80. 56±7. 14, P = 0. 009 ）. Conclusion TGF - β1/Smad2/3 signaling pathway plays an important regulation mechanism in osteoblastdifferentiation l^moted by low dose X -ray radiation.