PON1基因过表达对急性敌敌畏中毒小鼠膈肌细胞损伤的影响  

Effect of PON1 overexpression on mouse diaphragmatic muscle cells injury caused by acute dichlorvos poisoning

作　　者：吴斌[1] 王菲[1] 周觉[1] 候跃辉[1] 洪广亮[1] 赵光举[1] 葛赟[1] 刘瑶[1] 邱俏檬[1] 卢中秋[1] 

机构地区：[1]温州医科大学附属第一医院,325000

出　　处：《中华医学杂志》2015年第36期2955-2959,共5页National Medical Journal of China

基　　金：浙江省中医药重点学科建设计划（2012-XK-A28）,浙江省“十二五”高校重点学科建设计划,浙江省医药卫生平台骨干人才计划（2012RCB036）

摘　　要：目的 观察对氧磷酶1（PON1）基因过表达对急性敌敌畏中毒所致小鼠膈肌细胞损伤的影响.方法 常规培养小鼠膈肌细胞,转染过表达慢病毒载体,将细胞分为正常对照组,敌敌畏（DDVP）组,LV-GFP＋ DDVP组,LV-PON1＋ DDVP组,CCK-8法检测细胞存活率、流式细胞术检测细胞的凋亡情况、RT-PCR及Western印迹法检测小鼠膈肌细胞PON1和Nrf2mRNA及蛋白的表达情况、ELISA法检测细胞乙酰胆碱酯酶（AchE）、血红素氧合酶1（HO-1）、醌氧化还原酶1（NQO-1）水平,化学比色法检测细胞内超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活力及丙二醛（MDA）水平.结果 0、80、160、320、640 μmol/L DDVP诱导小鼠膈肌细胞24 h后,细胞的生存率分别为（100±3.82）％、（82.13 ±2.60）％、（53.57±5.05）％、（30.77±3.30）％、（14.20 ±2.19）％,呈浓度依赖性;以160μmol／L DDVP分别诱导小鼠膈肌细胞0、6、12、24 h后,细胞的存活率分别为（100.17±2.74）％、（76.13±6.01）％、（66.53±3.55）％、（53.57±5.05）％,呈时间依赖性（P＜0.05）.LV-PON1组细胞PON1蛋白表达高于空白对照组（0.370±0.015与0.232 ±0.004,0.197±0.015与0.037±0.003,P＜0.05）.DDVP诱导后,不同时间点LV-PON1组细胞存活率高于DDVP组（P＜0.05）.诱导24 h,LV-PON1组凋亡率及MDA含量低于DDVP组（P＜0.05）.LV-PON1组AchE水平、PON1和Nrf2蛋白表达水平、SOD和CAT、HO-1和NQO-1水平均高于DDVP组（P＜0.05）.结论 PON1基因过表达可以有效减轻DDVP对小鼠膈肌细胞AchE的抑制作用,可以诱导Nrf2的表达,发挥抗氧化作用,从而保护小鼠膈肌细胞.Objective To investigate the effect of paraoxonase1 （PON1） overexpression on mouse diaphragmatic muscle cells injury caused by acute dichlorvos poisoning.Methods Mouse diaphragmatic muscle cells were cultured routinely and infected with overexpression lentivirus.Cells were divided into normal control group,DDVP group,LV-GFP ＋ DDVP group,LV-PON1 ＋ DDVP group.Cell viability was determined by CCK-8 assay.Flow cytometry was used to detect cell apoptosis.The mRNA and protein expression of PON1 and Nrf2 in mouse diaphragmatic muscle cells was measured by RT-PCR and Western blot.Enzyme-linked immunosorbent assay was used to determine levels of acetyl cholinesterase （AchE）,heme oxygenase 1 （HO-1） and quinone oxidoreductase-1 （NQO-1） in mouse diaphragmatic muscle cells.The activity of superoxide dismutase （SOD） and catalase （CAT） as well as malondialdehyde （MDA） content in cells was measured by chemical colorimetry.Results After induced by 0,80,160,320,640μmol/L DDVP for 24 hours,the viability of mouse diaphragmatic muscle cells was （100 ± 3.82） %,（82.13 ±2.60）%,（53.57 ±5.05）%,（30.77 ±3.30）%,（14.20 ± 2.19）% respectively,changing in a concentration-dependent manner（P 〈 0.05）.After induced by 160 μmol/L DDVP for 0,6,12,24 hours,the viability of mouse diaphragmatic muscle cells was （100.17 ± 2.74） %,（76.13 ± 6.01） %,（66.53 ±3.55） %,（53.57 ± 5.05） %,changing in a time-dependent manner（P 〈 0.05）.The PON1 protein level in LV-PON1 group was higher than that of blank control group （0.370 ± 0.015 vs 0.232 ± 0.004,0.197 ± 0.015 vs 0.037 ±0.003,P 〈0.05）.The cell viability of LV-PON1 group is higher than that of DDVP group at different time point after induction of DDVP （P 〈 0.05）.After induced by DDVP for 24 hours,the cell apoptosis rate and MDA content in LV-PON1 group were lower than those of DDVP group（P 〈 0.05）.While levels of AchE,PON1 and Nrf2 protein expression,SOD and CAT,HO-1 and NQO-1 were higher than those of D

关 键 词：有机磷 敌敌畏 乙酰胆碱酯酶 氧化应激 

分 类 号：R595.4[医药卫生—内科学]