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目的通过分析氧化应激损伤的内耳毛细胞内游离Ca~(2+)浓度和钙调蛋白(CaM)表达水平,探讨感音性耳聋的氧化应激损伤机制。方法采用不同浓度(50μmol/L、100μmol/L、200μmol/L)双氧水染毒培养内耳毛细胞,24 h后检测细胞过氧化氢酶(CAT)活性,Fluo-3 AM荧光探针定量检测细胞内Ca~(2+)浓度,免疫荧光法分析CaM表达水平。结果氧化损伤的毛细胞CAT活性明显下降,随着染毒浓度的增加下降更明显(F=689.9,P<0.01);氧化损伤毛细胞游离Ca~(2+)浓度随着染毒浓度的增加而增加(F=716.107,P<0.01);氧化损伤的毛细胞CaM表达水平明显增加,50μmol/L和100μmol/L H_2O_2染毒组随浓度增加表达增强,但200μmol/L H_2O_2染毒组表达水平有所回落(F=732.727,P<0.01)。结论氧化应激损伤导致毛细胞内游离Ca~(2+)浓度和CaM表达增加诱发细胞凋亡是多类型感音性耳聋的重要发病机制,严重的氧化应激损伤可抑制细胞CaM表达。
OBJECTIVE: To investigate the mechanism of oxidative stress injury induced by sensorineural hearing loss by analyzing the expression of free Ca2 + and calmodulin (CaM) in the hair cells of the inner ear induced by oxidative stress. Methods The hair cells of the inner ear were cultured in different concentrations (50μmol / L, 100μmol / L, 200μmol / L) of H2O2. The activity of catalase (CAT) was measured 24 hours later. Fluo-3 AM Ca 2+ concentration and CaM expression level by immunofluorescence. Results Oxidatively damaged hair cells CAT activity decreased significantly, with the increase of the concentration decreased more significantly (F = 689.9, P <0.01); oxidative damage hair cells free Ca ~ (2+) concentration as the concentration increased (F = 716.107, P <0.01). The expression of CaM in oxidative damage hair cells increased significantly. The expression of CaM increased with the increase of concentration in 50μmol / L and 100μmol / L H 2 O 2 exposure groups, but the expression of CaM in 200μmol / L H 2 O 2 exposure group (F = 732.727, P <0.01). Conclusion Oxidative stress induced apoptosis in Ca2 + -induced intracellular free Ca2 + and apoptosis of Ca2 + -induced cells is an important pathogenesis of many types of sensorineural deafness. Serious oxidative stress can inhibit the expression of CaM.