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2922±291、4348±484、4660±375、5415±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。%Objective To investigate the effects of silencing peroxiredoxin-1 (Prx-1) gene on the proliferation, reac-tive 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-β1 group, 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 trans-fection and Prx-1 siRNA-453 was selected to be used later.Except for the control group, the other three groups were co-cultured 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-β1 stimulation.p-AKT expression was detected by Western blotting after 45 min of TGF-β1 stimulation.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-β1 group, negative transfection group and experimental group, and the levels of ROS were 2 922 ±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 con-trol group, TGF-β1 group, 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-β1 group 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 protein expression in the cells.
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