【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-β₁ 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-β₁ 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-β₁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-β₁/Smad信号通路激活有关。更多还原

【Abstract】 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-β₁intervention 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-β₁intervention 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-β₁/ Smad signaling pathway activation.更多还原