二甲双胍对Ⅰ型成骨不全小鼠间充质干细胞成骨分化的影响及疗效探讨

Study of effects of metformin on osteogenic differentiation of mesenchymal stem cells and bone tissue regeneration in type I osteogenesis imperfecta mice

目的研究二甲双胍对Ⅰ型成骨不全小鼠(oim)脂肪间充质干细胞(oimADSCs)向成骨细胞分化的影响并初步探讨二甲双胍在oim中的治疗效果。方法从oim中分离培养oimADSCs,通过观察细胞形态,碱性磷酸酶(ALP)染色和油红O染色对其分化能力进行鉴定;将oimADSCs分组进行诱导培养,通过观察ALP染色、检测ALP活性和实时荧光定量聚合酶链反应(qPCR)检测几种成骨分化相关分子的表达情况,分析二甲双胍对oimADSCs向成骨细胞分化的影响;利用微计算机断层扫描技术(Micro-CT)分析股骨的骨骼微观结构,观察二甲双胍在oim模型小鼠中的治疗效果。结果在二甲双胍处理oimADSCs后,ALP染色显著加深、ALP活性显著提高,骨钙素(Bglap)、Ⅰ型胶原(Col1a1)、Runt相关转录因子2(Runx2)以及骨形成蛋白2(BMP2)的mRNA表达水平升高;Micro-CT分析发现oim模型小鼠经过二甲双胍治疗后,股骨骨小梁的骨体积/总体积增加,股骨骨小梁数目增多、厚度增加。结论二甲双胍能够促进oimADSCs向成骨细胞分化,改善oim的骨骼生长情况。

Objective To evaluate the effect of metformin on the osteogenic differentiation of adipose derived mesenchymal stem cells of type I osteogenesis imperfecta mice(oimADSCs),and the therapeutic effect of metformin on bone tissue regeneration in oim mice.Methods The oimADSCs were isolated and cultured.Cell morphology observation,alkaline phosphatase staining and oil red O staining were used to validate the multiple differentiation potential of oimADSCs.After osteogenic induction,ALP staining and ALP activity were compared between groups.qPCR was applied to analyze the expression of osteogenic differentiation associated genes.Micro-CT was used to detect the femoral microstructure and analyze effects of metformin on bone regeneration in oim mice.Results Compared with the control group,ALP staining was more deeper and the ALP activity level was obviously increased in the osteogenesis+metformin group.The transcription levels of Bglap,Col1a1,Runx2 and BMP2 were significantly up-regulated.The micro-CT results revealed that after metformin treatment,BV/TV,trabecular number and Tb.Th of oim mice were increased.Conclusion Metformin can enhance osteogenic differentiation of oimADSCs in vitro and improve bone microstructure of oim mice in vivo.