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  

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

作　　者：荣书玲[1] 王庸晋[1] 王晓林[2] 赵俊青[3] 贺小峰[4] 胡耀东[1] 刘丽云[1] 

机构地区：[1]长治医学院附属和平医院心内科,山西长治046000 [2]长治医学院附属和平医院儿科,山西长治046000 [3]长治医学院附属和平医院康复科,山西长治046000 [4]长治医学院附属和平医院信息科,山西长治046000

出　　处：《中国病理生理杂志》2012年第10期1784-1790,共7页Chinese Journal of Pathophysiology

基　　金：山西省自然科学基金资助项目(No.2012011040-3);长治市科技计划项目(No.20111002;No.20123060)

摘　　要：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);Westernblotting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。AIM ： To observe the protective effect of human insulin - like growth factor 1 （ hlGF - 1 ） on rat skeletal myoblasts with ischemic/reperfusion injury. METHODS： Myoblasts were isolated from SD rats, cultured, purified, and transfected with plasmid pLghlGF - 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 trans- fected 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 hlGF - 1 in myoblasts transfected with pLghlGF - ISN was detected by immunoeytochemistry, RT- PCR and ELISA, but notin 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 deereased and bel - 2 mRNA significantly increased in IGF group eompared with GFP group （ P 〈 0. 05 ）. The results of Western blotting showed that the expression of easpase - 3 sig- nificantly decreased in IGF group compared with GFP group （P 〈 0. 05 ）. CONCLUSION： The transfeetion of hlGF- 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

关 键 词：胰岛素样生长因子1 成肌细胞 细胞增殖 细胞凋亡 

分 类 号：R363[医药卫生—病理学]