摘要
目的 探索谷氨酸 (glutamate,Glu)对离体耳蜗内毛细胞 (innerhaircells ,IHC)内钙信号的调控作用及其生理病理意义。方法 在激光共聚焦显微镜下用钙敏荧光探针Fluo 3作为指示剂 ,观察外源性谷氨酸对分离的 10个豚鼠耳蜗IHC胞内游离钙离子浓度 ([Ca2 + ]i)的影响。结果 分离好的正常IHC呈烧瓶形状 ,有明显的颈部 ,皮板上可观察到静纤毛 ,大球形的细胞体中间可见圆形的细胞核。形态完好的IHC大约存活 2h ,Fluo 3钙敏荧光探针染色后IHC胞体、胞核及表皮板有明显的染色梯度 ,表明游离Ca2 + 浓度从细胞核向细胞质逐渐减少。终浓度为 3.85 μmol L的Glu对游离IHC内 [Ca2 + ]i 有增高趋势 ,而对游离外毛细胞 (outerhaircells ,OHC)内 [Ca2 + ]i 浓度无影响。观察 10个IHC ,发现 9个 [Ca2 + ]i浓度增加 ,1个无变化 ;观察 10个OHC ,发现 7个 [Ca2 + ]i无变化 ,3个略有下降。当Glu浓度增高后 ,IHC内 [Ca2 + ]i 先是迅速升高 ,继而逐渐下降。IHC外形由烧瓶状逐渐变成球形 ,提示IHC水肿变性。结论 Glu可选择性调控IHC内 [Ca2 + ]i,而对OHC内 [Ca2 + ]i 无影响 ,而过量的Glu刺激 ,可造成IHC [Ca2 + ]i 的堆积 ,从而IHC水肿变性。
Objective To investigate the modulation effect of glutamate (Glu) on intracellular free Ca 2+ concentration([Ca 2+ ] i ) of inner hair cell (IHC). Methods Using the laser scanning confocal microscope(LSCM) , the exogenous Glu induced changes in [Ca 2+ ] i of isolated 10 IHCs and 10 OHCs of guinea pig cochlea were observed with fluo 3, a fluorescent probe for [Ca 2+ ] i. Results The IHCs were identified by their unique flask shape with a distinct neck and spherical base and a large spherical nucleus. In most cases, normal cell shapes could be maintained about two hours after isolation. The images of [Ca 2+ ] i from LSCM were similar to those from inverted microscopy. Fluorescence of Fluo 3 distributed in the isolated IHCs with brighter staining in the nucleus. In the presence of low concentration of Glu (3.85 μmol/L), there was an increase of [Ca 2+ ] i in IHCs, whereas no change in OHCs was found. Of the 10 IHCs, increases of [Ca 2+ ] i were observed in 9 and no change in 1. Of the 10 OHCs, 7 showed no [Ca 2+ ] i change and only 3 showed minor reduction of [Ca 2+ ] i. An increase of the Glu concentration (21.88 μmol/L)induced a corresponding increase of [Ca 2+ ] i in IHCs, but eventually resulted in a gradual decrease of [Ca 2+ ] i with a distortion of the normal shape, which indicated that the IHCs were degenerated and swelling. Conclusion These results suggested that exogenous Glu is capable of modulating [Ca 2+ ] i of IHC and may be act on autoreceptor in a positive feedback manner. Excessive Glu induced the accumulation of IHC [Ca 2+ ] i which finally resulted in the degeneration and edema of IHC and the reduction of IHC [Ca 2+ ] i.
出处
《中华耳鼻咽喉科杂志》
CSCD
北大核心
2001年第2期101-104,T004,共5页
Chinese Journal of Otorhinolaryngology
基金
国家自然科学基金!资助 ( 3 9870 797)
