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目的 探讨H2O2诱导的miR-21下调对MC3T3-E1细胞成骨分化的作用及机制.方法 取MC3T3-E1细胞系,培养传至第7代进行实验.取MC3T3-E1细胞,以不同浓度H2O2(0、40、80、160、320μmol/L)培养,经实时荧光定量PCR检测miR-21表达、MTS法检测细胞活性,选择H2O2最合适实验浓度.取MC3T3-E1细胞分为空白对照组(A组)、H2O2组(B组)、成骨诱导组(C组)、H2O2+成骨诱导组(D组),对应培养后实时荧光定量PCR检测miR-21表达以及成骨标志基因Runx2、骨桥蛋白(osteopontin,OPN)及Ⅰ型胶原蛋白(collagen type Ⅰ alpha 1,Col1a1)表达,Western blot检测磷酸酶与张力蛋白同源物(phosphatase and tensin homolog,PTEN)蛋白表达,茜素红染色观察细胞外钙基质沉积情况,分析H2O2对MC3T3-E1细胞成骨分化的影响.然后再取MC3T3-E1细胞,分为H2O2组(A1组)、H2O2+成骨诱导组(B1组)、H2O2+miR-21抑制剂+成骨诱导组(C1组)、H2O2+miR-21抑制剂阴性对照+成骨诱导组(D1组);以及H2O2组(A2组)、H2O2+成骨诱导组(B2组)、H2O2+siRNA-PTEN阴性对照+成骨诱导组(C2组)、H2O2+siRNA-PTEN+成骨诱导组(D2组);对应培养后检测成骨标志基因(Runx2、OPN、Col1a1)表达以及细胞外钙基质沉积情况,分析下调miR-21或沉默PTEN对细胞成骨分化的影响.结果 结合实时荧光定量PCR检测以及MTS法结果,选择160μmol/LH2O2进行实验.第1、2周B组miR-21相对表达量低于A组(P＜0.05),D组低于C组(p＜0.05);第2周C组PTEN蛋白相对表达量均低于A、D组(P＜0.05);第1、2周D组Runx2、OPN及Col1a1 mRNA相对表达量均低于C组(P＜0.05),茜素红染色显示D组钙基质沉积少于C组.C1组PTEN蛋白相对表达量高于D1组(P＜0.05);第1、2周B1、D1组Runx2、OPN mRNA相对表达量均高于C1组(P＜0.05),第2周B1、D1组Col1a1 mRNA均高于C1组(P＜0.05);茜素红染色显示C1组钙基质沉积少于B1、D1组.第1周D2组OPN、Col1a1 mRNA相对表达量高于B2、C2组(p＜0.05),第3周茜素红染色显示D2组钙基质沉积明显多于B2、C2组.结论 H2O2抑制MC3T3-E1成骨分化可能与miR-21下调有关.“,”Objective To explore the effect and mechanism ofmiR-21 down-regulated which was induced by H2O2 on osteogenic differentiation of MC3T3-E1 cells.Methods MC3T3-E1 cells were cultured and passaged,and the 7th generation cells were harvested to use in experiment.The MC3T3-E1 cells were treated with different concentrations (0,40,80,160,and 320 μmol/L) of H2O2.The expression of miR-21 was detected by real-time quantitative PCR (RT-PCR) and the cell viability was determined by MTS.Then the appropriate concentration of H2O2 was obtained.To analyze the effect of H2O2 on osteogenic differentiation of MC3T3-E1 cells,the MC3T3-E1 cells were divided into blank control group (group A),H2O2 group (group B),osteogenic induction group (group C),and H2O2+osteogenic induction group (group D).The expression of miR-21 and the osteogenesis related genes expressions of Runx2,osteopontin (OPN),and collagen type Ⅰ alpha 1 (Col1a1) were detected by RT-PCR.The expression of phosphatase and tensin homolog (PTEN) was detected by Western blot.The extracellular calcium deposition was detected by alizarin red staining.To analyze the effect on osteogenic differentiation of MC3T3-E1 cells after the transfection of miR-21 inhibitor and siRNA-PTEN,the MC3T3-E1 cells were divided into H2O2 group (group A1),H2O2+osteogenic induction group (group B1),H2O2+osteogenic induction+miR-21 inhibitor group (group C 1),and H2O2+osteogenic induction+miR-21 inhibitor negative control group (group D1);and H2O2 group (group A2),H2O2+osteogenic induction group (group B2),H2O2+osteogenicinduction+siRNA-PTEN negative control group (group C2),and H2O2+osteogenic induction+siRNA-PTEN group (group D2).The osteogenesis related genes were detected by RT-PCR and the extracellular calcium deposition was detected by alizarin red staining.Results The results of MTS and RT-PCR showed that the appropriate concentration of H2O2 was 160 μmol/L.The expression of miR-21 was significantly lower in group B than in group A at 1 and 2 weeks (P＜0.05).The expression of miR-21 was significantly lower in group D than in group C at 1 and 2 weeks (P＜0.05).The expression of PTEN protein was significantly lower in group C than in groups A and D (P＜0.05).The mRNA expressions of Runx2,OPN,and Col1a1 were significantly lower in group D than in group C at 1 and 2 weeks (P＜0.05).The extracellular calcium deposition in group D was obviously less than that in group C.The expression of PTEN protein was significantly higher in group C1 than in group D1 (P＜0.05).The mRNA expressions of Runx2 and OPN were significantly lower in group C1 than in groups B1 and D1 at 1 and 2 weeks (P＜0.05).The mRNA expression of Col1a1 was significantly lower in group C1 than in groups B1 and D1 at 2 weeks (P＜0.05).The extracellular calcium deposition in group C1 was obviously less than those in groups B1 and D1.The mRNA expressions of OPN and Col1a1 were significantly higher in group D2 than in groups B2 and C2 at 1 week (P＜0.05).The extracellular calcium deposition in group D2 was obviously more than those in groups B2 and C2.Conclusion H2O2 inhibits the osteogenic differentiation of MC3T3-E1 cells,which may be induced by down-regulating the expression of miR-21.