豚鼠D-半乳糖衰老模型耳蜗血管纹周细胞大电导钙激活钾离子通道变化的研究

Changes of BKCa on vascular striaepericytes of D-galactose-induced aging model in guinea pigs

目的探讨豚鼠D-半乳糖衰老模型耳蜗血管纹周细胞大电导钙激活钾离子通道(large conductance calcium-activated potassium channels,BKCa)的表达和功能变化及其与年龄相关性听力损失的关系。方法30只8周龄健康豚鼠按完全随机法分为三组,每组10只:D-半乳糖衰老模型组,每天颈部皮下注射D-半乳糖(500 mg/kg),连续6周;生理盐水对照组:每天颈部皮下注射与衰老模型组同体积生理盐水,连续6周;空白对照组:不做任何处理。造模结束后检测各组豚鼠听性脑干反应(auditory brainstem response,ABR)阈值;通过免疫荧光技术检测各组豚鼠耳蜗血管纹周细胞上BKCa的表达情况;利用膜片钳技术检测周细胞电流密度及BKCa电流的变化情况。数据采用GraphPad Prism软件进行统计分析。结果与生理盐水组和对照组相比,衰老模型组豚鼠ABR阈值升高且Ⅰ波振幅明显降低,差异均具有统计学意义(P值均<0.01)。与对照组相比,衰老模型组豚鼠血管纹周细胞上BKCa的表达明显减少(1.00±0.08 vs 0.27±0.03,差异有统计学意义,P<0.01),周细胞电流密度和BKCa净电流值亦明显减小(差异均具有统计学意义,P值均<0.01)。结论D-半乳糖可成功诱导豚鼠衰老模型,其耳蜗血管纹周细胞上BKCa表达下降、净电流值减小,BKCa表达和功能的变化可能与年龄相关性听力损失有关。

Objective The aging model of guinea pigs induced by D-galactose was set up to investigate the changes of BKCa expression and function on cochlear pericytes and their relationship with age-related hearing loss.Methods Thirty healthy 8-week-old guinea pigs were randomly divided into three groups,with 10 in each group:D-galactose aging model group,subcutaneous injection of D-galactose(500 mg/kg)daily for 6 weeks;saline control group,the same amount of saline was injected into the neck of the aging model group for 6 weeks;the blank control group,no treatment was performed.The threshold of auditory brainstem response(ABR)was detected.The content of BKCa in the perivascular cells of the guinea pig cochlear cells was detected by immunofluorescence technique.The changes of peripheral current density and BKCa current were detected by patch clamp technique.The data were analyzed by GraphPad Prism software.Results Compared with the saline group and the control group,the ABR threshold and the amplitude of the wave I were significantly decreased in the aging model group,and the difference was statistically significant(P<0.01).Compared with the control group,the expression of BKCa in the vascular pericytes of guinea pigs in the aging model group was significantly reduced(1.00±0.08 vs 0.27±0.03,the difference was statistically significant P<0.01),and the cell current density and BKCa net current value were also significantly reduced with statistically significant(P<0.01).Conclusions D-galactose can successfully induce guinea pig aging model,in which BKCa expression decreases and net current value decreases in pericytes of cochlear striavascularis,and changes in BKCa expression and function may be related to age-related hearing loss.