中文摘要：

目的观察硫氧还原蛋白过氧化物酶1（Prx-1）基因沉默对转化生长因子β1（TGF-β1）诱导肺成纤维细胞增殖、活性氧簇（ROS）水平及磷酸化AKT（p-AKT）蛋白表达的影响。方法体外培养肺成纤维细胞MRC-5,并分为对照组、TGF-β1组、阴性转染组和实验组。阴性转染组和实验组分别通过脂质体Lipofectamine2000转染阴性对照siRNA及设计好的3个Prx-1 siRNA（Prx-1 siRNA-209、Prx-1 siRNA-289、Prx-1 siRNA-453）,培养48 h,real-time PCR法检测Prx-1 mRNA,选取转染Prx-1 siRNA-453的细胞用于后续实验。除对照组外,其余三组给予TGF-β1（5μg/L）刺激。TGF-β1刺激24 h后,MTT实验观察细胞增殖情况,2,7-二氯荧光素二乙酸（DCFH-DA）实验检测ROS水平;TGF-β1刺激45min后,Western blotting法检测p-AKT蛋白。结果对照组、TGF-β1组、阴性转染组、实验组OD值分别为0.56±0.07、0.81±0.10、0.88±0.18、1.16±0.18,ROS水平分别为2 922±291、4 348±484、4 660±375、5 415±436;实验组细胞增殖情况及ROS水平与其余三组相比,P均〈0.01;TGF-β1组、阴性转染组与对照组相比,P均〈0.01。对照组、TGF-β1组、阴性转染组、实验组细胞中p-AKT蛋白相对表达量分别为0.45±0.05、0.60±0.07、0.57±0.07、0.77±0.09;实验组与其余三组相比,P均〈0.01;TGF-β1组与对照组相比,P〈0.01。结论Prx-1基因沉默可增强TGF-β1对肺成纤维细胞的增殖诱导作用,上调细胞内ROS水平和p-AKT蛋白表达。

英文摘要：

Objective To investigate the effects of silencing peroxiredoxin-1（ Prx-1） gene on the proliferation,reactive oxygen species（ ROS） and phosphorylated AKT（ p-AKT） protein expression of pulmonary fibroblasts induced by TGF-β1. Methods The cultured MRC-5 pulmonary fibroblasts were randomly divided into 4 groups： control group,TGF-β1group,negative transfection group and experimental group. The Prx-1 siRNA 3 sequences of Prx-1 siRNA（ Prx-1 siRNA-209,Prx-1 siRNA-289,Prx-1 siRNA-453） were transfected into the negative transfection group and experimental group by Lipofectamine2000,respectively. After culturing for 48 h,Prx-1 mRNA was detected by real-time PCR to evaluate transfection and Prx-1 siRNA-453 was selected to be used later. Except for the control group,the other three groups were cocultured with TGF-β1（ 5 μg/L）. Cell proliferation was detected by MTT assay and reactive oxygen species（ ROS） level was detected by DCFH-DA assay after 24 h of TGF-β1stimulation. p-AKT expression was detected by Western blotting after45 min of TGF-β1stimulation. Results The OD was respectively 0. 56 ± 0. 07,0. 81 ± 0. 10,0. 88 ± 0. 18 and 1. 16 ±0. 18 in the control group,TGF-β1group,negative transfection group and experimental group,and the levels of ROS were2 922 ± 291,4 348 ± 484,4 660 ± 375 and 5 415 ± 436,respectively. Significant difference were found in the levels of ROS and OD between the experimental group and the other three groups（ all P〈0. 01）. The p-AKT expression of the control group,TGF-β1group,negative transfection group and experimental group was 0. 45 ± 0. 05,0. 60 ± 0. 07,0. 57 ± 0. 07 and 0. 77 ± 0. 09,respectively. The expression of p-AKT in the experimental group was the highest in all groups（ all P〈0. 01）. The p-AKT expression of the TGF-β1group was higher than that of the control group（ P〈0. 01）. Conclusion Silencing Prx-1 gene can enhance the proliferation of pulmonary fibroblasts induced by TGF-β1,and up-regulate the level of ROS and p-AKT prot