siRNA抑制GPX1表达对新霉素诱导HEI-OC1细胞凋亡的影响  

Effect of Inhibition GPX1 by siRNA on Apoptosis of HEI-OC1 Cells Induced by Neomycin

作　　者：陈放 王雪峰 Chen Fang;Wang Xuefeng(Otolarygology Head and Neck Surgery,the First Affiliated Hospital of Jinzhou Medical University,Jinzhou,121000,China)

机构地区：[1]锦州医科大学附属第一医院耳鼻咽喉头颈外科,锦州121000

出　　处：《听力学及言语疾病杂志》2022年第4期416-421,共6页Journal of Audiology and Speech Pathology

摘　　要：目的分析抑制谷胱甘肽过氧化物酶1(GPX1)对氨基糖苷类抗生素损伤HEI-OC1细胞凋亡的影响。方法首先将HEI-OC1细胞培养后采用免疫荧光法观察GPX1的表达;然后采用CCK8法计算不同浓度新霉素处理HEI-OC1细胞后细胞活力及其半抑制浓度(IC50);再设立对照组和新霉素组,对照组不进行任何处置,新霉素组给予新霉素处理,采用免疫荧光染色、qRT-PCR和Western blot法研究新霉素给药后GPX1的表达;第四步设立空白对照组、阴性对照组和实验组,空白对照组不进行任何处置,阴性对照组转染无意义的siRNA,实验组转染siRNA-GPX1,使用免疫荧光染色、qRT-PCR和Western blot法检测siRNA转染效率,CCK8法观察三组细胞活力变化;转染成功后后两组给予新霉素处理,采用免疫荧光法检测三组细胞活力,并采用免疫荧光染色、TUNEL染色和Western blot法检测三组细胞凋亡状况。结果在HEI-OC1细胞可见中GPX1表达,并且新霉素损伤后HEI-OC1细胞活力随着新霉素的浓度升高而降低,新霉素半抑制浓度为10 mM;新霉素组细胞的GPX1蛋白、mRNA表达水平(分别为0.21±0.07,0.35±0.08)均较对照组(分别为0.38±0.02,1.00±0.16)降低(均为P<0.05)。转染siRNA-GPX1后实验组的GPX1蛋白和mRNA表达水平相对于阴性对照组明显受到抑制,但实验组的细胞活力相对于阴性对照组没有明显变化(P>0.05);给予新霉素损伤后,与阴性对照组细胞计数(301.33±38.76个)比较,实验组的细胞计数(183.67±18.58个)明显减少(P<0.01);实验组TUNEL阳性细胞比例(22.76%±1.66%)高于阴性对照组(10.84%±3.38%)(P<0.01);实验组中Cleaved Caspase-3阳性细胞比例(47.20%±9.22%)高于阴性对照组(14.24%±1.45%)(P<0.001);相对于阴性对照组,实验组BCL-2的蛋白水平下降(P<0.05),BAX的相对蛋白水平升高(P<0.001)。结论抑制氨基糖苷类抗生素损伤毛细胞的GPX1表达可导致HEI-OCI细胞凋亡程度加重,表明GPX1可能具有抗凋亡的作用;GPX1�Objective To investigate the effect of inhibiting glutathione peroxidase 1(GPX1)on the apoptosis of HEI-OC1 cells injured by aminoglycosamine antibiotics.Methods The first step was to culture HEI-OC1 cells and observe the expression of GPX1 by immunoassay.In the second step,the CCK8 method was used to calculate the cell viability and the half-inhibitory concentration(IC50)of HEI-OC1 cells treated with different concentrations of neomycin.The third step was to establish a control group and a neomycin group,the control group was given no treatment,and the experimental group was treated with neomycin.Immunofluorescence,qRT-PCR and Western blot methods were used to study the expression of GPX1 after neomycin administration.The fourth step was to set up the blank control group,the siRNA-control group and the siRNA-GPX1 group.The blank control group was not treated,the siRNA-control group was transfected with meaningless siRNA,and siRNA-GPX1 group was transfected with siRNA-GPX1.Immunofluorescence,qRT-PCR and Western blot methods were used to detect siRNA transfection efficiency,and CCK8 methods were used to observe the changes in cell viability of the three groups.Finally,after successful transfection,neomycin was given.The immunofluorescence method was used to detect the viability of the three groups of cells,and the immunofluorescence,TUNEL staining and Western blot methods were used to detect the apoptosis levels of the three groups of cells.Results GPX1 was expressed in HEI-OC1 cells.The viability of HEI-OC1 cells after neomycin injury decreased with the increase of the concentration of neomycin,and the half-inhibitory concentration of neomycin was 10 mM.After neomycin administration,the expression levels of GPX1 protein and mRNA in the neomycin group decreased.The protein and mRNA expression levels of GPX1 were significantly inhibited after transfection of siRNA-GPX1(P<0.001).However,the cell viability of the experimental group did not change significantly compared with the negative control group(P>0.05).After ne

关 键 词：谷胱甘肽过氧化物酶1 新霉素 耳蜗毛细胞 凋亡 

分 类 号：R764.3[医药卫生—耳鼻咽喉科]