摘要
目的研究促凋亡基因Par-4沉默对氧化应激导致的肺泡上皮细胞凋亡的影响。方法培养人肺泡Ⅱ型上皮细胞A549细胞。利用过氧化氢诱导细胞凋亡。利用小RNA干扰（siRNA）技术，靶向诱导Par4基因沉默。设立正常对照组（常规培养细胞）、过氧化氢组（1．0mmol／L过氧化氢处理细胞）、过氧化氢＋Par-4-siRNA组（1．0mmol／L过氧化氢和Par4-siRNA转染的细胞）。设非抑制序列转染的对照组。流式细胞术测定各组细胞凋亡百分率。Western blot检测促凋亡基因Smac蛋白表达量。凝胶迁移率改变试验测定转录因子E2F1的DNA结合力。比色法检测Caspase-3酶活性。实验结果采用单因素方差分析（F检验和q检验）。结果过氧化氢＋Par4-siRNA组细胞凋亡百分率（29．74±2．3）％显著低于过氧化氢组（54．24±4．1）％，q=8．91，P〈0．01。Par-4-siRNA的转染显著抑制过氧化氢诱导的肺泡上皮细胞中Smac蛋白表达、E2F1DNA结合力和caspase-3活性上调。结论采用siRNA诱导Par-4基因沉默，可减少氧化应激导致的肺泡上皮细胞凋亡。其分子机制可能和抑制Smac蛋白表达、抑制E2F1 DNA结合力和抑制caspase-3活性有关。
Objective To investigate the effects of Par-4 gene silence on hydrogen peroxide-induced apoptosis in alveolar epithelial cells. Method The alveolar epithelial cells A549 were cultured and exposed to hydrogen peroxide. The siRNA sequences targeted Par-4 gene was chemically synthesized and transfected to A549 cells with or without the exposure of hydrogen peroxide. The cells were divided into normal control groups, hydrogen peroxide-treated group（The cells were treated with 0.1 mmol/L hydrogen peroxide） , hydrogen peroxide and Par-4-siRNA-treated group（ The cells were treated with 0.1 mmol/L hydrogen peroxide after transfection of Par-4-siRNA ） , Non-specific DNA sequence transfection control group. The apoptosis of A549 cells was quantified by flow cytometry. The expression of Smac protein was detected by Western blot. Electrophoretic mobility shift assay was applied for evaluating the change of E2F1 DNA binding activity. Relative activity of Caspase-3 was detected by clolorimetric assay. Results The percent of apoptotic cells in hydrogen peroxide and Par-4-siRNA-treated group was （29.7 ± 2.3 ） %, which was significantly lower than that of hydrogen peroxide-treated group [ （54.2 ±4.1 ） % , q = 8.91, P 〈 0.01 ） ]. Par-4 siRNA could significantly suppress the increase of Smac protein, E2F1 DNA binding activity and caspase-3 activity induced by hydrogen peroxide in A549 cells. Conclusions Par-4 gene silence induced by siRNA might inhibit the apoptosis of alveolar epithelial cells, which might be resulted from suppression of the up-regulation of Smac gene expression, E2F1 DNA binding activity and caspase-3 activity.
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2011年第3期269-272,共4页
Chinese Journal of Emergency Medicine
基金
江苏省卫生厅面上科研课题（H200901）
