硫酸新霉素对新生大鼠离体培养耳蜗毛细胞的毒性作用研究

目的观察体外培养的新生大鼠耳蜗Corti器经不同浓度硫酸新霉素处理后,各回毛细胞损伤情况。方法取新生大鼠完整耳蜗基底膜,体外培养12小时,存活贴壁后加药处理。对照组10个样本;3个实验组各10个样本,分别用终浓度为1mmol/L、2mmol/L、4mmol/L硫酸新霉素处理24小时。进行MyosinⅦa免疫荧光组织化学染色,在共聚焦显微镜下观察各回毛细胞缺失情况。分别选取10个图片进行毛细胞计数（每张图片截取100μm计数毛细胞个数）,并利用CHISS软件进行统计分析。对照组除不加入硫酸新霉素外,其他培养条件均与实验组相同。结果硫酸新霉素对离体培养的新生大鼠耳蜗毛细胞具有损伤效应,且外毛细胞对硫酸新霉素的毒性作用比内毛细胞敏感。加入硫酸新霉素后,耳蜗毛细胞损失从底回外毛细胞开始,随着药物浓度增加,逐渐累及到中回,最后顶回受累,且损失程度随着浓度的递增而加重,以外毛细胞为甚。单纯体外培养的耳蜗毛细胞没有缺失。经完全随机设计方差分析可得：顶回正常组内毛细胞数量与新霉素1mmol/L组、2mmol/L组无统计学差异,与新霉素4mmol/L组有统计学差异;中、底回正常组内毛细胞数量与新霉素1mmol/L组、新霉素2mmol/L组、新霉素4mmol/L组均有统计学差异;顶回正常组外毛细胞数量与新霉素2mmol/L与4mmol/L组有统计学差异;中、底回正常组外毛细胞数量与新霉素1mmol/L组、新霉素2mmol/L组、新霉素4mmol/L组均有统计学差异。结论新生大鼠体外培养的耳蜗毛细胞随着硫酸新霉素药物浓度的增加损失越严重,且从底回向顶回发展,此与活体动物实验的损害规律基本相同,因此可作为耳毒性药物损伤后毛细胞再生研究的体外模型。

Effects of Neomycin Sulfate on Cultured Neonatal Rat Cochlear Hair cells at Different Concentrations

Objective To study injury effects of neomycin sulfate at different concentrations on cultured neonatal rat Cochlear hair cells. Methods The organ of Corti in the whole cochlea from neonatal (P0) SD rats was isolated and cultured for 12 hours. Neomycin sulfate of different final concentrations was added to surviving culture media. Forty samples were randomly selected into a control and 3 test groups (n = 10 in each group). Samples in the test groups were treated with neomycin sulfate at 1 mmol/L, 2 mmol/L or 4 mmol/L for 24 hours. They were then prepared for confocal microscope examination via immunofluorescence staining of Myosin Ⅶ-a. The number of hair cells within a 100 μmsegment was counted on 10 arbitrarily selected images and compared statistically using the CHISS software. Samples in the control group received no neomycin treatment. Results When compared to the control group, the number of inner hair cells in the apical turn showed statistically significant differences in only samples treated with 4 mmol/L neomycin sulfate. However, the number of inner hair cells in the middle and basal turns was statistically different from the control group in all neomycin treated samples. The number of outer hair cells in the apical turn was statistically different from the control group in samples treated with 2 mmol/L and 4 mmol/L neomycin sulfate. The difference in middle and basal turns was of statistical significance for all three neomycin sulfate concentrations. Conclusion The results indicate toxic effects on Cochlear hair cells by neomycin sulfate, with outer hair cells being more sensitive than inner hair cells. The damage appears to start from the basal turn. Increasing neomycin concentration not only increases the severity of hair cell damage, but increasingly involves the middle and finally the apical turn, especially outer hair cells. No hair cell damage was seen in the control group. The ototoxic effects of neomycin sulfate to ochlear cultures are similar to those seen on cochlear hair cells reported in in vivo studies. This can serve as a model for in vitro hair cell regeneration research.