氧化应激损伤对内耳毛细胞内游离钙离子和钙调蛋白表达的影响

The effect of oxidative stress on intracellular free Ca^(2+) and CaM expression in hair cells

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 analyze concentration of intracellular free Ca2＋and the expression level of calmodulin（CaM） in hair cells with oxidative stress,and to investigate the mechanism of sensory deafness caused by oxidative stress. Methods Hair cells were cultured with different concentrations（50 μmol/L,100 μmol/L,200 μmol/L） of hydrogen peroxide for 24 h,the catalase（CAT） activity were tested. Fluo-3 AM fluorescent probe were used to detect intracellular free Ca2＋concentrations quantitatively. The expression levels of CaM were detected by immunofluorescence analysis. Results The CAT activity decreased obviously in hair cells with oxidative damage. The group with higher concentrations of hydrogen peroxide decreased more significantly（F= 689. 9,P〈0. 01）. The concentrations of intracellular free Ca2＋increased obviously in hair cells with oxidative damage and those in the groups with higher concentrations of hydrogen peroxide increased more significantly（F = 716. 107,P〈 0. 01）. The expression levels of CaM increased significantly in hair cells with oxidative damage; and those in 50 μmol/L and 100 μmol/L exposure group increased along with the increscent of concentrations of hydrogen peroxide. However,the expression levels of CaM in 200 μmol/L exposure group decreased（F = 732. 727,P〈0. 01）. Conclusion Oxidative stress increased the concentrations of intracellular free Ca2＋and the expression levels of CaM in the hair cells,which maybe an important pathogenesis of sensory deafness,although more serious oxidative stress inhibit the expression level of CaM in hair cells.