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

陈放;王雪峰;

1:锦州医科大学附属第一医院耳鼻咽喉头颈外科

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可能成为预防氨基糖苷诱导耳毒性的新潜在靶标。
关键词
谷胱甘肽过氧化物酶1;新霉素;耳蜗毛细胞;凋亡
基金项目(Foundation):
作者
陈放;王雪峰;
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陈放王雪峰