摘要
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 study the important role of Runt-related transcription factor 2（Runx2） in the Hedgehog-induced terminal osteoblast differentiation.Methods MC3T3-E1 cells were cultured and treated with Sonic Hedgehog（Shh）.The cells were divided into 4 groups： control group,Shh treatment group（Shh group）,Runx2 siRNA treatment group（Runx2 siRNA group） and Runx2 siRNA ＋ Shh treatment group（Runx2 siRNA ＋ Shh group）.Runx2 expression was inhibited by Runx2 siRNA transfection.The expressions of Runx2 and osteoblastic factors [alkaline phosphatase（ALP）,osteocakin（OC）,bone sialoprotein（Bsp）,Col 1a1 and Osx）] mRNA and protein were detected by real-time polymerase chain reaction（PCR） and Western blot.Alkaline phosphatase（ALP） staining was performed to detect ALP protein expression.Results The results of real-time PCR and Western blot showed that Shh increased the mRNA and protein expression of MC3T3-E1 osteoblastic factors（ALP,OC,Bsp,Col 1a1 and Osx）.The ALP staining density in the Shh group was higher than that in the control group.Inhibition of Runx2 expression significantly decreased the mRNA and protein expression of osteoblastic factors（ALP,OC,Bsp,Col 1a1 and Osx） and ALP staining density in MC3T3-E1 cells,indicated that Shh failed to promote osteoblastic differentiation after Runx2 expression was inhibited.Conclusion It was demonstrated that Runx2 mediated Hedgehog could promote osteoblast terminal differentiation,and the Hedgehog promote the terminal mature differentiation of osteoblast through up-regulating Runx2 expression.
出处
《生物医学工程与临床》
CAS
2013年第4期384-388,共5页
Biomedical Engineering and Clinical Medicine
