急性分离大鼠皮层神经元方法与体会.docVIP

急性分离大鼠皮层神经元方法与体会 　　[摘要] 目的：建立急性分离大鼠皮层神经元的方法。方法：切取活脑组织切片，经Protease酶解消化、孵育后，采用酶加机械分离法制备10~16 d鼠龄的大鼠皮层神经元。作形态学观察，用全细胞膜片钳技术记录其电生理学特性。结果：分离出的神经元倒置相差显微镜下形态保持良好，有较长的轴突；用膜片钳技术证实，80%以上细胞可以记录出电活动，其保存了主要的离子通道活性。结论：成功建立了一种经济、简便的适用于膜片钳技术的大鼠皮层神经元急性分离方法。 　　[关键词] 皮层神经元；急性分离；膜片钳；离子通道 　　[中图分类号] R965 [文献标识码] A [文章编号] 1673-7210（2011）06（c）-041-03 　　 　　Study on the separating the cortical neurons of rats by acute enzyme separation method 　　WANG Zhuang1, LIU Fu2, JI Hui3, TIAN Hua4* 　　1.Office of Student Work of Adult and Further Education College of Qiqihar Medical University, Qiqihar 161006, China; 2.Office of Basic Medical College of Qiqihar Medical University, Qiqihar 161006, China;3.Department of Anatomy of Qiqihar Medical University, Qiqihar 161006, China;4.Department of Pharmacology of Qiqihar Medical University, Qiqihar 161006, China 　　[Abstract] Objective: To establish a method which is described for separating the rat cortical neurons. Methods: Cuting a slice of live brain sections, after Protease enzymatic hydrolysis digestion, incubation, the cortical neurons were obtained by a combination of mechanical and enzymatic means from 10-16 day-old rats. Observing by morphology, their electrophysiological properties were recorded by whole-cell patch-clamp recording technique. Results: the difference of the inverted cortical neurons which were obtained by separation kept good under microscope. They each had a long axon. Proved by the patch-clamp recording technique, more than 80% cells could be recorded their electric activity, which preserved the ion channel activity. Conclusion: An economic and simple method was established, which is described for separating the rat cortical neurons by the patch-clamp recording technique. 　　[Key words] Cortical neurons; Acute isolation; Patch clamp; Ion channel 　　 　　近年来，研究细胞膜离子通道活动的主要方法是膜片钳技术，而获得活力好的神经元便成为应用膜片钳技术的重要先决条件。???多离子通道研究是需要单个分散的神经元，人工培养的神经细胞易受培养环境的影响，自身特性改变较大，而急性分离的细胞却能相对保持完好的生理特性[1-3]。笔者结合文献并加以改进，摸索出一种简便易行的适用于全细胞膜片钳研究的大鼠顶叶皮层神经