低剂量哇巴因对螺旋神经节细胞损伤的保护作用

Low dose of ouabain protects injury of spiral ganglion neurons in vitro

目的:探讨低剂量哇巴因对B27剥夺诱发的螺旋神经节细胞损伤的保护作用,并初步探讨其保护机制。方法:螺旋神经节细胞培养第7天开始分组实验,设B27剥夺损伤组(损伤组)、B27剥夺加10nmol/L哇巴因保护组(保护组)和正常培养对照组(对照组)。各组继续培养48h后用FITC标记的Annexin-V和碘化丙啶双染试剂盒染色,用流式细胞仪检测各组螺旋神经节细胞损伤情况。各组螺旋神经节细胞分别于加药后6、12h两个时间点用免疫细胞化学方法检测细胞内Bcl-2表达水平。另外,应用螺旋神经节组织块分组培养48h后光学显微镜下观察轴(树)突生长情况。结果:流式细胞仪检测结果显示,保护组螺旋神经节细胞损伤率为(9.0±1.3)%,显著低于损伤组(32.8±2.4)%,与对照组(6.3±0.3)%相比无明显差异;免疫细胞化学结果显示,保护组螺旋神经节细胞6h点Bcl-2水平较损伤组和对照组增加。螺旋神经节组织块培养显示,保护组轴(树)突生长明显优于损伤组。结论:低浓度哇巴因对B27剥夺诱发的螺旋神经节细胞损伤具有保护作用,此作用可能通过上调螺旋神经节细胞内Bcl-2表达水平来实现。

Objective：To evaluate the protective effect of low dose ouabain on injury of cultured spiral ganglion neurons evoked by trophic factors deprived and to explore the mechanism. Method：Spiral ganglion neurons were cultured in vitro for 7 days, and then exposed to Neurobasal medium＋B27 supplement,Neurobasal medium only or Neurobasal medium＋10 nmol/L ouabain, respectively. After 48 h exposed to different medium, spiral ganglion neurons were stained by FITC labeled Annexin-V and PI, then apoptosis index were calculated using fluorescent microscope and flow cytometry, respectively. In addition, spiral ganglion tissues were cultured for 48 h to evaluate dendrite growth of spiral ganglion neurons in each group. Immunocytochemistry were performed to detect the level of Bcl-2 in each group at 6 h and 12 h. Result：Spiral ganglion neurons exposed to Neurobasal medium ＋ 10 nmol/L ouabain have a similar apoptosis index compare with that of Neurobasal medium＋ B27 supplement, but a much lower apoptosis index than that of Neurobasal medium only. In addition, dendrite growth of spiral ganglion neurons exposed to Neurobasal medium ＋ 10 nmol/L ouabain was much longer than that of Neurobasal medium only. Bcl-2 level increased in spiral ganglion neurons exposed to Neurobasal medium ＋ 10 nmol/L ouabain at 6 h. Conclusion：Low dose of ouabain protects injury of spiral ganglion neurons evoked by trophic factors deprived in vitro. This effect may mediated by increasing the level of Bcl-2.