SIRT1调控BMSCs成骨分化与H型血管生成促进骨质疏松骨缺损愈合的研究

SIRT1 Regulates Osteogenic Differentiation of BMSCs and H-type Vessels Formation to Promote Healing of Osteoporotic Bone Defects

目的:探究沉默信息调节因子1(silent information regulator 1,SIRT1)通过调控骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化与H型血管生成对老年骨质疏松状态下骨缺损愈合的作用,并探讨其具体分子机制。方法:利用免疫组织化学染色检测小鼠骨组织中SIRT1表达的增龄性改变。使用SIRT1激活剂SRT1720与抑制剂EX527作用于BMSCs,实时荧光定量逆转录聚合酶链反应(quantitative real-time PCR,RT-qPCR)与Western blot检测成骨标志物以及促H型血管形成因子的表达变化,免疫荧光染色与Western blot检测上述过程中Wnt/连环蛋白β(β-catenin)信号通路变化。构建老年骨质疏松骨缺损模型,SRT1720与EX527作用的同时,分别给予Wnt信号通路抑制剂XAV939与激活剂CHIR-99021,Micro-CT、苏木精-伊红(hematoxylin-eosin staining,HE)/Masson染色与免疫荧光染色检测各组骨缺损区域新骨形成与H型血管含量差异。结果:小鼠骨组织中SIRT1表达呈现增龄性下调趋势。SIRT1能够促进成骨因子碱性磷酸酶(alkaline phosphatase,ALP)、矮小相关转录因子2(Runt-related transcription factor 2,RUNX2)和促H型血管形成因子slit同源物3(slit homolog 3,SLIT3)、血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,并促进老年骨质疏松骨缺损区域内H型血管形成与骨再生,且上述作用由Wnt/β-catenin信号通路介导。结论:SIRT1能够通过调控Wnt/β-catenin信号通路促进BMSCs成骨分化与H型血管生成,进而促进老年骨质疏松小鼠的骨缺损愈合。

Objective:To investigate the role of silent information regulator 1(SIRT1)on H-type vessels regeneration and bone defect healing in senile osteoporotic state by regulating osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and to explore its specific molecular mechanism.Methods:Immunohistochemical staining was used to detect age-related changes in SIRT1 expression in mouse bone tissue.BMSCs were treated with SIRT1 activator SRT1720 and inhibitor EX527,and the changes in the expression of osteogenic markers and H-type angiogenic factors were detected by RT-qPCR and Western Blot,and the changes in the Wnt/β-catenin signaling pathway in the above process were detected by immunofluorescence staining and Western Blot.A model of osteoporotic bone defects was constructed in the elderly,and SRT1720 and EX527 were administered with XAV939,an inhibitor of Wnt signaling pathway,and CHIR-99021,an activator of Wnt signaling pathway,respectively.The differences in the formation of new bone and H-vessel content of the defects were detected by Micro-CT,HE/Masson staining,and immunofluorescent staining in the respective groups.Results:The expression of SIRT1 showed an age-increasing down-regulation trend in mouse bone tissues.SIRT1 was able to promote the expression of alkaline phosphatase(ALP),Runt-related transcription factor 2(RUNX2),and the pro-H-type angiogenic factors slit homolog 3(SLIT3)and vascular endothelial growth factor(VEGF),and able to promote the H-type angiogenesis and bone regeneration in the region of osteoporotic bone defects in old age,which was mediated by the Wnt/β-catenin signaling pathway.Conclusion:SIRT1 can promote the osteogenic differentiation and H-type angiogenesis of BMSCs by regulating the Wnt/β-catenin signaling pathway,which in turn promotes the healing of bone defects in the osteoporotic state of aged mice.