摘要
目的建立大鼠耳蜗血管纹缘细胞氧化性损伤的体外模型。方法在体外培养的大鼠耳蜗血管纹缘细胞中加入过氧化氢(H2O2),观察细胞形态结构变化;采用CCK-8(cell counting kit-8)法检测200、300、400、600、800μmol/LH202作用0.5、1、2.4、16、24h对血管纹缘细胞活性的影响;检测不同浓度H2O2作用2h后血管纹缘细胞脂质过氧化产物丙二醛含量的变化;利用碘化丙锭染色流式细胞仪测定细胞的凋亡率;通过免疫印迹法(Western blot)检测凋亡因子半胱氨酸天冬氨酸蛋白酶3(caspase-3)活化片段cleaved—caspase-3的表达。结果H2O2作用后血管纹缘细胞出现核固缩、边缘化,胞质浓缩,被膜包裹、隆起,产生凋亡;随着H2O2:浓度的增加、作用时间的延长,缘细胞活性降低;200μmoVL的H2O2作用2h,即可诱导缘细胞凋亡率升高,差异具有统计学意义(P〈0.05);cleaved.caspase-3在正常缘细胞呈微弱表达,H2O2作用后cleaved—caspase-3表达增强,并随H2O2浓度的增高而增强,但当H20:达到600μmol/L时,表达开始减弱,800μmol/L时仅见微弱表达。结论利用H2O2可成功建立耳蜗血管纹缘细胞氧化性损伤的体外模型,caspase-3的激活参与了缘细胞的氧化损伤过程。
Objective To set up the oxidative stress experimental model of rat cochlea with stria vascularis marginal ceils injury induced by hydrogen peroxide in vitro. Methods Cultured marginal cells of rat were treated by 200,300,400,600 and 800μmol/L hydrogen peroxide (H2O2 ) for 0. 5,1,2,4,16 and 24 hours, respectively. Cell viability was assessed by the CCK-8 assay. The content of the lipid peroxidation production malondialdehyde(MDA) were detected in H2O2 induced marginal cells injury with different concentration H2O2. Apoptosis was assessed by flow cytometry by propidium iodium staining. The expression of the eleaved-caspase-3 was assessed by Western blot. Results Being exposed to H2O2, marginal cells displayed nuclear pyknosis and margination, cytoplasmic condensation, cell shrinkage and formation of membrane and bounded apoptotic bodies. A time-dependent and dose-dependent decrease of cellular viability was detected with the treatment of H2O2. Cellular maleic dialdehyde was generated in proportion to the concentration of H2O2 at 2 hours and the number of apoptotic cells increased significantly (P 〈 0. 05 ) . Western blot showed the expression of the cleaved-caspase-3 increased when 200 μmol/L,300 μmol/L and 400μmol/L H2O2 treated cultured marginal cells. Thereafter the expression of the cleaved-easpase-3 decreased with 600 μmol/L H2O2 and with 800 μmol/L H2 O2 the expression of cleaved-caspase-3 was weak. Conclusions The findings indicated that the experimental model can be established successfully using cultured cells exposed to H2 O2 and activation of caspase-3 is associated with hydrogen peroxide induced rat marginal cells the oxidative stress injury.
出处
《中华耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2008年第11期835-839,共5页
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
基金
国家自然科学基金(30730094)
关键词
血管纹
细胞
培养的
过氧化氢
氧化性应激
细胞凋亡
Stria vascularis
Cells, cultured
Hydrogen peroxide
Oxidative stress
Apoptosis