【摘要】 目的探讨胰岛素样生长因子-1(IGF-1)对大鼠心肌细胞凋亡保护作用的基因调控机制。方法体外培养新生大鼠心肌细胞,10nmol/L IGF-1刺激的同时,分别加入磷脂酰肌醇-3激酶(PI3K)、细胞外信号调节激酶(ERK)1/2和Raf-1 3条通路抑制剂(20μmol/L),通过RT-PCR及Western blotting方法观察IGF-1调节基本转录元件结合蛋白(BTEB)的基因表达及其通路调控。100μmol/L H2O2处理诱导心肌细胞凋亡,通过DNA梯度分析、Annexin V-FITC/PI双染色法、Caspase-3活性测定、Hoechest33258染色法观察用BTEB特异性siRNA人为下调BTEB基因表达后对心肌细胞凋亡的影响。结果大鼠心肌细胞经IGF-1刺激60min后,BTEB mRNA和蛋白表达均明显下降;与对照组相比,加入ERK1/2通路抑制剂PD98059组BTEB的mRNA和蛋白表达均明显增高(P<0.01);H2O2诱导的大鼠心肌细胞于下调BTEB表达后,DNA片段化改善,心肌细胞凋亡率下降(P<0.05),Caspase-3活性降低(P<0.05),凋亡小体减少,与IGF-1的抗心肌细胞凋亡效果相似。结论 IGF-1可以通过ERK1/2通路下调转录因子BTEB基因表达而发挥抗心肌细胞凋亡的作用。更多还原

【Abstract】 Objective To investigate gene regulation mechanism of insulin-like growth factor-1( IGF-1) antiapoptotic effect on rat cardiomyocytes. Methods Primary neonatal rat cardiomyocytes( NRCMs) were cultured in vitro,IGF-1( 10 nmol / L) was added with different signal transduction pathway inhibitors [phosphatidylinositol 3-kinase( PI3K),extracellular regulated kinase( ERK) 1 /2 and Raf-1]respectively( 20μmol / L). The gene expression of basic transcription element binding protein( BTEB) was detected by RT-PCR and Western blotting,by which the pathway of IGF-1 downregulated BTEB gene expression was judged. NRCMs were treated with 100 umol / L hydrogen peroxide( H2O2) to induce apoptosis. BTEB specific siRNA was transfected into the cells by Lipofectamine 2000. Myocardial cells apoptosis was detected by DNA-ladder analysis,Annexin V-FITC / PI dual staining,Caspase-3 activity assay and Hoechst33258 staining.Results The mRNA and protein expression levels of BTEB gene in NRCMs were down-regulated significantly after IGF-1had stimulated for 60 minutes. Compared with control groups,BTEB mRNA and protein expression in ERK1 /2 pathway inhibitor PD98059 group was significantly higher( P < 0. 01). The apoptosis of NRCMs was induced by H2O2. Artificiallyinhibited BTEB gene expression with BTEB specific siRNA,BTEB mRNA and protein expression decreased obviously( P <0. 05). Compared with control group,the apoptotic rates of NRCMs induced by H2O2 in IGF-1 group and BTEB specific siRNA groups were declined( all P < 0. 05),decreased Caspase-3 activity( all P < 0. 05),attenuated DNA fragmentation and reduced apoptotic bodies were also observed in these groups. The anti-apoptotic effect of BTEB gene silencing on NRCMs was similar with that of IGF-1 treatment. Conclusion IGF-1 protects cardiomyocytes from apoptosis by downregulating transcription factor BTEB through ERK1 /2 pathway.更多还原