胰岛素受体底物1基因沉默对3T3-L1细胞CCAAT增强子结合蛋白α、过氧化物酶体增殖物激活受体γ表达的影响

摘要

目的 研究胰岛素受体底物1(IRS-I)沉默对3T3-L1前脂肪细胞分化关键分子CCAAT增强子结合蛋白α(C/EBPα)、过氧化物酶体增殖物激活受体γ(PPARγ) mRNA及蛋白表达的影响,并观察IRS-1沉默后前脂肪细胞分化为成熟脂肪细胞能力的改变,探讨胰岛素受体底物(IRSs)是否作为C/EBPα 、PPAR-γ的上游调节信号在前脂肪细胞分化中起到重要的调节作用.方法 合成4条IRS-I shRNA,Western blotting筛选出沉默效率最高的1条.3T3-L1前脂肪细胞分为IRS-1沉默组和对照组.IRS-1沉默组细胞用沉默效率最高的IRS-1 shRNA质粒转染3T3-LI前脂肪细胞,沉默细胞中的IRS-1分子；对照组采用无意义IRS-1 shRNA质粒转染3T3-L1前脂肪细胞.转染24h后诱导其分化成熟,分化72 h后检测促进前脂肪细胞分化的关键分子C/EBPα、PPARγ的表达.继续诱导分化至第7天,油红O染色观察IRS-1沉默后前脂肪细胞分化为成熟脂肪细胞的比例,检测其分化能力的改变.结果 与对照组相比,IRS-1沉默组在诱导分化72 h后,Real-time PCR结果显示3T3-L1前脂肪细胞中C/EBPα mRNA、PPARγ mRNA表达明显下降(Pa＜0.05),Western blotting结果显示C/EBPα、PPARγ蛋白水平也同样显著下降(Pa＜0.05)；细胞继续分化至第7天,油红O染色显示,IRS-1沉默组前脂肪细胞分化成熟的比例与对照组比较显著降低.结论 胰岛素可能通过IRS-1刺激C/EBPα、PPARγ的表达促进前脂肪组织的分化.%Objective To examine the influence of insulin receptor substrate 1 (IRS - 1) gene silence on the mRNA and protein expression of CCAAT enhancer binding protein a( C/EBPa) and peroxisome proliferator - activated receptor -y( PPAR-y) in 3T3 - LI preadipocyte cell, to observe the changes of its differentiation ability into mature cell when IRS - 1 gene was silenced, and explore whether IRS - 1 was a key upstream regulatory molecule of C/EBPa and PPAR-y in preadipocyte cell differentiation. Methods Four recombination plasmids of IRS - 1 shRNA were constructed and the most effective one was selected by Western blotting. The cells infected with the most effective IRS -1 shRNA plasmid were grouped as IRS - 1 silence group and cells incubated with non - sense IRS - 1 shRNA as control group. The cells were induced into mature cell after 24 hours' incubation with transfection. The expression levels of mRNA and protein of C/EBPa and PPAR-y were tested by Western blotting and real-time PCR.and the differentiation level was examined by red oil,respectively,at 72 h and 7 d after the differentiation. Results Compared with the control group, both mRNA and protein expression of C/EBPa and PPAR7 in IRS - 1 silence group had significant decrease at 72 h after the cells differentiation (Pt <0.05) ,and the ratio of mature cells which was tested by red oil reduced obviously in IRS -1 silence group at 7 d after the differentiation. Conclusion Insulin can stimulate the expression of C/EBPa and PPAR7 by IRS - 1 in order to promote preadipocyte cells differentiation.