骨形态发生蛋白2对人尿源性干细胞骨向分化的影响

The influence of bone morphogenetic protein 2 on osteogenic differentiation of human urine-derived stem cells

目的探讨骨形态发生蛋白2(BMP2)对人尿源性干细胞(hUSCs)骨向分化的影响,并研究其相关机制。方法体外提取hUSCs,利用流式细胞仪检测表型CD29、CD90、CD73、CD44、CD34及CD45;利用10-7~10-5mol/L浓度的BMP2干预hUSCs,Western blot检测骨性标志物碱性磷酸酶(ALP)和Runt相关转录因子2(Runx2);利用基因沉默干扰腺苷酸活化蛋白激酶(si-AMPK),并检测骨性标志物表达。建立大鼠颅骨缺损模型,通过将细胞附着在骨织工程修复支架β-磷酸三钙(β-TCP)植入颅骨缺损模型,1个月后micro CT检测新骨体积和新骨厚度并利用病理学观察新骨结构。结果流式细胞仪检测结果显示,hUSCs高表达CD29、CD90、CD73、CD44,低表达CD34、CD45;并且BMP2在10-6mol/L对hUSCs具有最佳促进成骨效果。Western blot显示,si-AMPK能有效抑制BMP2诱导的骨性标志物ALP和Runx2表达。体内实验结果显示,BMP2能有效促进新骨形成,而si-AMPK能有效抑制新骨形成。结论BMP2在体内外均能有效促进hUSCs骨向分化,可能是通过激活AMPK途径发挥作用。

Objective To explore the effect of bone morphogenetic protein 2(BMP2)on the osteogenic differentiation of human urine-derived stem cells(hUSCs)and its related mechanism.Methods The hUSCs were extracted in vitro,and the phenotypes CD29,CD90,CD73,CD44,CD34 and CD45 were detected by flow cytometry.The bone markers alkaline phosphatase(ALP)and Runt-associated transcription factor 2(Runx2)were detected by Western blot after the intervention of hUSCs with BMP2 concentration of 10-7~10-5mol/L.Gene silencing was used to interfere with AMP-activated protein kinase(si-AMPK),and bone marker expression was detected.The skull defect model of rats was established,and the skull defect model was implanted with cells attached to the bone tissue engineering repair scaffoldβ-tricalcium phosphate(β-TCP).After 1 month,the new bone volume and thickness were detected by micro CT,and the new bone structure was observed by pathology.Results The results of flow cytometry showed that the hUSCs showed high expression of CD29,CD90,CD73,CD44 and low expression of CD34,CD45.In addition,BMP2 had the best osteogenic effect on hUSCs at 10-6mol/L.Western blot showed that si-AMPK could effectively inhibit the expression of bone markers ALP and Runx2 induced by BMP2.In vivo results showed that BMP2 could effectively promote new bone formation,while si-AMPK could effectively inhibit new bone formation.Conclusions BMP2 can effectively promote bone differentiation of hUSCs in vivo and in vitro,possibly play a role through activating AMPK pathway.