人胰岛素样生长因子1基因转染对大鼠骨骼肌成肌细胞缺血再灌注损伤的影响

目的: 探讨人胰岛素样生长因子1(hIGF-1)基因转染对大鼠骨骼肌成肌细胞体外缺血再灌注损伤的影响及机制。方法: 分别用pLghIGF-1SN质粒(IGF组)和对照质粒pLgGFPSN(GFP 组)转染大鼠成肌细胞。未转染的成肌细胞(control组)作为细胞对照。转染后用免疫细胞化学、RT-PCR及ELISA检测基因表达。转染后3~14 d检测各组细胞的扩增率。转染的细胞接受缺血再灌注损伤后7 d,TUNEL法检测各组细胞的凋亡百分数,RT-PCR 检测各组细胞的bax和bcl-2 mRNA的表达,Western blotting检测各组细胞caspase-3的表达。结果: 基因转染后免疫细胞化学和RT-PCR 检测发现IGF组细胞有hIGF-1表达,GFP组和control组细胞未见hIGF-1表达;IGF组细胞上清中可检测到hIGF-1的蛋白表达,control组和GFP组细胞上清中未检测到hIGF-1蛋白表达。转染后14 d,IGF组细胞的扩增率显著大于control组和GFP组(P< 0.05);细胞经缺血再灌注损伤后,TUNEL染色检测结果显示:与GFP组相比,IGF组细胞的凋亡百分比显著降低(P< 0.05);RT-PCR 检测结果显示:与GFP组相比,IGF组细胞的bax mRNA表达显著降低,bcl-2 mRNA的表达显著增加(P< 0.05);Western blotting检测结果显示:与GFP组相比,IGF组细胞caspase-3蛋白表达显著降低。结论: IGF-1基因转染可增加成肌细胞的抗凋亡能力,其机制可能和降低Bax和caspase-3表达,增加Bcl-2的表达有关。

Protective effect of human insulin-like growth factor 1 gene transfection on rat skeletal myoblasts with ischemic/reperfusion injury

AIM: To observe the protective effect of human insulin-like growth factor 1 (hIGF-1) on rat skeletal myoblasts with ischemic/reperfusion injury. METHODS: Myoblasts were isolated from SD rats, cultured, purified, and transfected with plasmid pLghIGF-1SN or pLgGFPSN. The myoblasts were divided into insulin-like growth factor (IGF) group (myoblasts transfected with pLghIGF-1SN), green fluorescent protein (GFP) group (myoblasts transfected with pLgGFPSN), and control group (untransfected myoblasts). The expression of hIGF-1 in myoblasts was investigated by immunocytochemistry, RT-PCR and ELISA. The proliferation rate of myoblasts 14 days after transfection was detected. To observe the protective effect of IGF-1 gene on skeletal myoblasts with ischemic/reperfusion injury 7 days after transfection, the apoptotic myoblasts were detected by the method of in situ TdT-mediated dUTP nick end labeling (TUNEL). The expression of bax and bcl-2 mRNA was detected by RT-PCR, and the expression of caspase-3 was determined by Western blotting. RESULTS: The expression of hIGF-1 in myoblasts transfected with pLghIGF-1SN was detected by immunocytochemistry, RT-PCR and ELISA, but not in myoblasts transfected by pLgGFPSN and untransfected myoblasts. The proliferation rate of myoblasts in IGF group was higher than that in other groups (P<0.05). The results of RT-PCR showed that the expression of bax mRNA significantly decreased and bcl-2 mRNA significantly increased in IGF group compared with GFP group (P<0.05). The results of Western blotting showed that the expression of caspase-3 significantly decreased in IGF group compared with GFP group (P<0.05). CONCLUSION: The transfection of hIGF-1 gene mediated by a retroviral vector produces a protective effect in rat skeletal myoblasts with ischemic/reperfusion injury. The mechanisms may be associated with down-regulating the expression of Bax and caspase-3 and up-regulating Bcl-2 expression.