在爪蟾卵母细胞中表达大鼠耳蜗核钾通道的方法学研究

Study on the methodolgy of potassium channel expression in Xenopus oocytes by messenger RNA from rat cochlear nucleus

目的研究在爪蟾卵母细胞中表达的大鼠耳蜗核电压依赖性离子通道的特性，为揭示感音神经性聋听觉中枢离子通道的变化奠定方法学基础。方法采用异硫氰酸胍－苯酚氯仿法和美国新磁珠技术，从５０只大鼠耳蜗核提取多聚腺嘌呤ｍＲＮＡ，注入非洲爪蟾卵母细胞表达有功能的离子通道，并利用电压钳方法记录电压依赖性的离子通道电流。结果在注射大鼠耳蜗核ｍＲＮＡ的１６个卵母细胞膜上记录到电压依赖性的瞬间外向型钾离子通道电流，其电流最大幅度的平均值为３６４±４２ｎＡ。结论本实验建立的模型在听觉中枢的生理、病理及药理学研究中起到一定的作用。

Objective To study the properties of voltage dependent ion channels expressed in Xenopus oocytes by mRNA from rat cochlear nucleus and to lay a methodological foundation for studying the changes of ion channels in auditory centre with sensorineural hearing loss. Methods Poly (A) + mRNAs isolated from the rat cochlear nucleus by acid guanidinum thiocyanate phenol chloroform (AGPC) method and Promega′s new magnesphere technique were microinjected into Xenopus laevis oocytes to express functional ion channels. Voltage clamp technique was used to record the current of voltage dependent ion channels. Results A voltage dependent ion channel was detected from oocytes injected with rat cochlear nucleus mRNA. The maximum amplitude of the outward current was 364±42 nA ( n =16). It was identified as transient outward potassium channel . Conclusion The model set up by the test may play an important role in studying the physiology、pathology and pharmacology of auditory centre.