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目的探讨Runt-related transcription factor 2(Runx2)在Hedgehog促进成骨细胞晚期分化过程中的重要介导作用。方法培养MC3T3-E1细胞,以Sonic Hedgehog(Shh)处理细胞。将细胞分为4组:对照组;Shh组;Runx2 siRNA组;Runx2 siRNA+Shh组。应用siRNA技术沉默细胞内Runx2基因,real-time PCR法和Western blot方法检测细胞内Runx2和碱性磷酸酶(ALP)、骨钙素(OC)、骨涎蛋白(Bsp)、Col1a1、Osx成骨因子mRNA和其蛋白的表达水平,ALP染色方法检测细胞内ALP的蛋白表达强度。结果 Real-time PCR法和Western blot方法检测结果表明,Shh明显提高MC3T3-E1细胞ALP、OC、Bsp、Col 1a1、Osx成骨因子mRNA、蛋白的表达。ALP染色表明,Shh组ALP的染色强度高于对照组。沉默Runx2基因后,real-time PCR法和Western blot方法检测结果 ,与对照组比,Runx2 siRNA组、Runx2 siRNA+Shh组MC3T3-E1细胞内ALP、OC、Bsp、Col 1a1、Osx成骨因子mRNA、蛋白的表达明显降低;ALP染色表明,ALP染色强度明显下降;表明Shh失去其促进成骨细胞分化的作用。结论 Runx2介导Hedgehog促进成骨细胞的晚期分化,Hedgehog可通过上调Runx2的表达,提高其功能,促进成骨细胞的晚期成熟分化。
Objective To investigate the important role of Rux-related transcription factor 2 (Runx2) in Hedgehog-induced osteoblast differentiation. Methods MC3T3-E1 cells were cultured and treated with Sonic Hedgehog (Shh). The cells were divided into 4 groups: control group; Shh group; Runx2 siRNA group; Runx2 siRNA + Shh group. Runx2 gene was silenced by siRNA. The expression of Runx2, alkaline phosphatase (ALP), osteocalcin (OC), bone sialoprotein (Bsp), Col1a1 and Osx in osteoblasts were detected by real-time PCR and Western blot. Factor mRNA and its protein expression level, ALP staining method to detect intracellular ALP protein expression intensity. Results Real-time PCR and Western blot showed that Shh significantly increased the mRNA and protein expression of osteogenic factor (ALP, OC, Bsp, Col 1a1 and Osx) in MC3T3-E1 cells. ALP staining showed that the staining intensity of ALP in Shh group was higher than that in control group. After silencing the Runx2 gene, real-time PCR and Western blot results showed that compared with the control group, the expression of ALP, OC, Bsp, Col 1a1, Osx mRNA in Runx2 siRNA group and Runx2 siRNA + Shh group , The protein expression was significantly reduced; ALP staining showed that ALP staining intensity decreased significantly; Shh lost its role in promoting osteoblast differentiation. Conclusion Runx2 mediates the late differentiation of osteoblasts mediated by Hedgehog. Hedgehog can enhance the function of Runx2 by up-regulating the expression of Runx2, and promote the late mature differentiation of osteoblasts.