Objective To explore the ability and mechanism of insulin-like growth factor l (IGF-1) induced bone mesenchymal stem cells ( BMSCs) differentiation into cardiomyocyte-like cells ( CLCs).Methods BMSCs were isolated and purified in vitro.BMSCs were treated with control medium and 15 ng/ml IGF-I for 3,7, 14 and 21 d, respectively. The expression of Troponin-T ( TNT), Troponin-I (TNI) and pIGF-1R were c~etected by immunocytochemistry and Westem blot.In another experimental setting,BMSCs were treated with control medium and 15 ng/ml IGF-l,ICF-1 antaS;onist I-Ome AG538 (300 nmol/L) and 300 nmol/L I-Ome AG538 + 15 ng/ml IGF-1 for 3 t0 48 h,respectively.Phosphorylation status of ERKl/2 and AKT,the two downstream mediators of mitogen-activated protein kinase ( MAPK) kinase and phosphatidylinositol 3-kinase ( PBK) pathways,were detected by immunocytochemistry and Yvestem blot.Results After 3 t0 21 d exposrue to IGF-1, the expression of pIGF1R,TNT and TNI were significandy higher in 'IGF-1 group than those in control group,pIGF-IR peaked 14 d (all P ＜0.05).After 3 and 6 h treatment,the ratio of pAKT/AKT(0.17 ± 0.03)and pERK1/2/ERK1/2(0.06 ± 0.03)were significantly downregulated in I-Ome AG538 group compared to control group (1.00 ±0.05) (all P ＜ 0.05).The ratio of pAKT/AKT(1.00 ± 0.07) and pERK1/2/ERK1/2 (1.00 ± 0.09) were significantly upregulated in IGF-1 group compared to control group (0.72 ± 0.05) (all P ＜ 0.05),but the ratio of pAKT/AKT(0.31 ± 0.10)and pERK1/2/ERK1/2 (0.39 ±0.04) were significantly downregulated in I-Ome AG538 group compared to control group (0.63 ± 0.05) (all P ＜ 0.05),the value of gray scale of TNT (195.06 ± 5.98) and TNI (198.32 ± 3.46) in I-Ome AG538 + IGF-1 group were significantly upregnlated than that in IGF-1 group for TNT (188.70 ± 5.35) and TNI (176.10 ± 4.96) (all P ＜ 0.05).Conclusions IGF-1 could induce BMSCs differentiation into CLCs in vitro by activating MAPK and PI3K signaling pathways.%目的 研究胰岛素样生长因子1(IGF-1)诱导骨髓间充质干细胞(BMSC)分化为心肌样细胞的能力及其机制.方法 分离大鼠BMSC,并在体外培养纯化.实验分为两个部分:第一部分,IGF-1诱导BMSC分化为心肌样细胞的实验研究,分为培养基对照组(对照组)和15 ng/ml的IGF-1诱导组(IGF-1组),连续培养21 d,分别在第3、7、14和21天采用免疫细胞化学染色法和Western blot法检测BMSC中磷酸化(p) IGF-1受体(R)、心肌特异性肌钙蛋白T(TNT)及肌钙蛋白I(TNI)的表达.第二部分为IGF-1R激酶阻断剂I-OMe AG538阻断IGF-1诱导BMSC向心肌样细胞分化的实验研究,分为培养基对照组(对照组)、15 ng/ml IGF-1诱导组(IGF-1组)、300 nmol/L I-OMe AG538阻断组(I-OMe AG538组)及300 nmol/L I-OMe AG538阻断+15 ng/ml IGF-1诱导组(I-OMe AG538+ IGF-1组).免疫细胞化学法和Western blot法检测BMSC中TNT及TNI、MAPK和PI3K信号通路的蛋白表达水平.结果 IGF-1诱导BMSC分化的实验结果显示IGF-1组诱导后,TNT、TNI的表达均呈阳性,pIGF-1R的表达量也明显高于对照组(上述因子均未检测到),以第14天为最高(P＜0.05).I-OMeAG538作用BMSC 3和6h后,I-OMe AG538组pERK1/2/ERK1/2比值分别为0.17±0.03和0.06±0.03,均显著低于对照组的1.00±0.05(P均＜0.05).IGF-1组pAKT/AKT和pERK1/2ERK1/2的比值分别为1.00±0.07和1.00±0.09均显著高于对照组的0.72±0.05(P均＜0.05),而I-OMe AG538组pAKT/AKT和pERK1/2 ERK1/2的比值分别为0.31±0.10和0.39±0.04均显著低于对照组的0.63±0.05(P均＜0.05).I-OMe AG538+ IGF-1组心肌样细胞的TNT和TNI蛋白表达的灰度值分别为195.06±5.98和198.32±3.46均显著高于IGF-1组的188.70±5.35和176,10±4.96(P均＜0.05).IGF-1组IGF诱导BMSC 3、7、14和21 d后,其TNT、TNI和pIGF-1R的蛋白表达水平均高于对照组,且以第14天为最高(P均＜0.05).结论 IGF-1诱导BMSC分化为心肌样细胞的最佳诱导时间是14 d,诱导机制可能与MAPK和PI3K信号通路有关.
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