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背景:许多离子通道研究需要单个分散的神经元。人工原代培养的神经细胞受环境的影响,自身特性改变较大,而急性分离的神经元却能相对保持完好的生理特性。目的:建立急性分离尾核神经元的方法,用膜片钳技术研究尾核神经元离子通道及其信号转到机制,利于深入了解尾核的功能。设计、时间及地点:以细胞为对象的观察实验,于2008-02/12在三峡大学医学院实验中心完成。材料:新生7~10dWistar乳鼠12只,雌雄不限。方法:取7~10d的大鼠,采用酶和机械分离法制备分散的单个尾核神经元,利用全细胞膜片钳技术记录电压依赖性钙电流。主要观察指标:用全细胞膜片钳急性分离大鼠尾核神经元的形态学视察和电生理特性。结果:用链蛋白酶(Protease)消化及机械分离法,急性分离的新生大鼠尾核神经元,表面光洁,胞膜完整,有较长的突起,形态和生理特性良好。利用急性分离的新生大鼠尾核神经元观察L-钙离子通道电流,所记录的电学参数在正常生理范围内,较好地保存电压依赖性钙离子通道活性。结论:分离出的神经元形态正常,有较长的轴突;保存了主要的离子通道活性,成功建立了一种适用于膜片钳技术的大鼠尾核神经元急性分离方法。
Background: Many ion channel studies require single, discrete neurons. Artificial primary cultured nerve cells are greatly affected by the environment, and their characteristics are greatly changed. However, acutely isolated neurons can relatively maintain intact physiological characteristics. OBJECTIVE: To establish a method for the acute isolation of caudate nucleus neurons by using patch-clamp technique to study the ion channels in the caudate nucleus and their signal transduction mechanism, which helps to understand the function of caudate nucleus. DESIGN, TIME AND SETTING: The cell-based observation experiment was performed at the Experimental Center of the Three Gorges University Medical College between February 2008 and December 12. MATERIALS: Twelve Newborn Wistar suckling mice aged 7-10 days, both male and female. Methods: The isolated single caudate nucleus neurons were prepared by enzymatic and mechanical separation methods and the voltage-dependent calcium current was recorded by whole-cell patch-clamp technique. MAIN OUTCOME MEASURES: Morphological examination and electrophysiological characteristics of acutely isolated rat caudate nucleus neurons using whole-cell patch clamp technique. Results: The neurons of neonatal rat caudate nucleus acutely isolated by protease digestion and mechanical separation had smooth surface, complete cell membrane, long protuberance and good morphological and physiological characteristics. L-Ca 2+ channel currents were observed using acutely isolated neonatal rat caudate nucleus neurons. The recorded electrical parameters were within the normal physiological range and the voltage-dependent calcium channel activity was well preserved. CONCLUSION: The isolated neurons are normal and have long axons. The main ion channel activity is preserved and a new method of acute separation of rat caudate nucleus suitable for patch-clamp technique has been successfully established.