Prx-1基因沉默对TGF-β_1诱导肺成纤维细胞增殖、ROS水平、p-AKT蛋白表达的影响

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...

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Bibliographic Details
Published in山东医药 Vol. 56; no. 7; pp. 17 - 19
Main Author 刘宝欣 刘英宇 魏中秋 梁婷婷 范玉磊 杨方 孙影
Format Magazine Article
LanguageChinese
Published 2016
Subjects
基因沉默
活性氧簇
硫氧还原蛋白过氧化物酶1
细胞增殖
肺成纤维细胞
蛋白激酶B
转化生长因子β1
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Summary: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。
Bibliography:LIU Baoxin, LIU Yingyu, WEI Zhongqiu, LIANG Tingting, FAN Yulei, YANG Fang, SUN Ying （ 1 Tangshan Workers＇ Hospital Affzliated to North China University of Sciencg and Technology., Tangshan 063000, China）
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
ISSN:1002-266X