KLF4基因沉默大鼠肝星状细胞中Smad 2、3、7 mRNA及蛋白表达观察  

Effects of silencing KLF4 on Smad2,3 and 7 mRNA and protein in rat hepatic stellate cells

作　　者：李涛[1] 牛丽娟[2] 刘文宣[1] 杨磊[1] 李曼[1] 曹丹丹[1] 刘殿武[1] 

机构地区：[1]河北医科大学公共卫生学院,石家庄050017 [2]石家庄市第三医院

出　　处：《山东医药》2016年第31期24-27,共4页Shandong Medical Journal

基　　金：河北省卫生和计划生育委员会医学科学研究重点课题计划项目(20150627)

摘　　要：目的观察Kruppel样因子4(KLF4)基因沉默对大鼠肝星状细胞HSC-T6中Smad 2、3、7 mRNA及蛋白表达的影响。方法设计并构建针对大鼠KLF4基因的3个干扰质粒(p GPU6-1、p GPU6-2、p GPU6-3),将质粒转染HSC-T6细胞,检测KLF4 mRNA及蛋白,筛选出沉默效果最好的重组质粒用于后续实验。将HSC-T6细胞分为A、B、C、D组,A组常规培养细胞;B组在培养液中加入TGF-β_1 50 ng/m L;C组转染重组质粒p GPU6-3;D组转染干扰质粒p GPU6-3 24 h后,再加入TGF-β_1 50 ng/m L。采用real-time PCR法检测各组细胞中的Smad2、Smad3、Smad7mRNA。采用Western blotting法检测各组细胞中的Smad2、Smad3、Smad7蛋白和磷酸化Smad2、3(p-Smad2、3)蛋白。结果 B、D组细胞中Smad2、Smad3、Smad7 mRNA相对表达量高于A组,C组细胞中Smad7 mRNA相对表达量高于A组(P均<0.05)。C组、D组细胞中p-Smad2、p-Smad3蛋白相对表达量低于A组,Smad7蛋白相对表达量高于A组(P均<0.05)。B组细胞中Smad2、p-Smad2、Smad3、p-Smad3、Smad7蛋白相对表达量均高于A组(P均<0.05)。结论 KLF4基因沉默后,HSC-T6细胞(包括被TGF-β_1激活的HSC-T6)中p-Smad2、p-Smad3蛋白表达下调,Smad7 mRNA及蛋白表达上调;抑制KLF4基因表达可能有抗纤维化作用,作用机制与抑制TGF-β_1/Smad信号通路激活有关。Objective To detect the effects of silencing Kruppel-like factor 4（ KLF4） gene on the Smad 2,3 and 7mRNA and protein expression of rat hepatic stellate cells HSC-T6. Methods Three plasmids（ p GPU6-1,p GPU6-2 and p GPU6-3） targeting KLF4 mRNA were designed. The chosen p GPU6-3 with the best silencing effects was infected into rat HSCT6 cells and experiments were performed. HSC-T6 cells were divided into four groups： groups A,B,C and D. In group A,cells were cultured in DMEM mediun. In group B,cells were cultured in DMEM medium and TGF-β_1intervention solution to a final concentration of 50 ng / m L. In group C,HSC-T6 was transfected with interfering plasmid p GPU6-3. In group D,50 ng / m L TGF-β_1intervention solution was added after the transfection of interfering plamid p GPU6-3. Real-time PCR was used to measure the mRNA expression of Smad2,Smad3 and Smad7 in HSC-T6 cells. The protein expression of Smad2,phosphorylated p-Smad2,Smad3,phosphorylated p-Smad3 and Smad7 was detected in HSCs-T6 cells by Western blotting. Results The relative expression levels of Smad2,Smad3 and Smad7 mRNA in the groups B and D were higher than those in the group A,and the relative expression level of Smad7 mRNA in the group C was higher than that in the group A（ all P〈0. 05）. The relative expression levels of p-Smad2 and p-Smad3 protein in groups C and D were lower than those in the group A,and the relative expression level of Smad7 protein was higher than that in the group A（ all P〈0. 05）. The relative expression levels of Smad2,p-Smad2,Smad3,p-Smad3 and Smad7 protein in group B were higher than those in the group A（ all P〈0. 05）.Conclusions After KLF4 silencing,the expression of Smad2 and Smad3 in HSC-T6 cells was down-regulated and the protein expression of Smad7 was up-regulated. Inhibiting the KLF4 gene expression may have anti-fibrosis effect,and its mechanism may be related with the inhibition of TGF-β_1/ Smad signaling pathway activation.

关 键 词：Kruppel样因子4基因 人肝星状细胞 转化生长因子β1 SMAD 基因沉默 肝纤维化 

分 类 号：R575.2[医药卫生—消化系统]