中国修复重建外科杂志

中国修复重建外科杂志

H2O2 下调 miR-21 对 MC3T3-E1 细胞成骨分化影响的研究

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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、320 μmol/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 160 μmol/L H2O2 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。

ObjectiveTo explore the effect and mechanism of miR-21 down-regulated which was induced by H2O2 on osteogenic differentiation of MC3T3-E1 cells.MethodsMC3T3-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 C1), and H2O2+osteogenic induction+miR-21 inhibitor negative control group (group D1); and H2O2 group (group A2), H2O2+osteogenic induction group (group B2), H2O2+osteogenic induction+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.ResultsThe 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.ConclusionH2O2 inhibits the osteogenic differentiation of MC3T3-E1 cells, which may be induced by down-regulating the expression of miR-21.

关键词: H2O2; 氧化应激; miR-21; 成骨分化; MC3T3-E1 细胞

Key words: H2O2; oxidative stress; miR-21; osteogenic differentiation; MC3T3-E1 cells

引用本文: 彭建强, 黄年盛, 黄胜, 李亮平, 凌泽民, 靳松, 黄爱军, 林昆, 邹学农. H2O2 下调 miR-21 对 MC3T3-E1 细胞成骨分化影响的研究 . 中国修复重建外科杂志, 2018, 32(3): 276-284. doi: 10.7507/1002-1892.201707030 复制

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