果蝇晚三龄幼虫脑神经元的快速分离与鉴定  

Acute Isolation and Identification of Cerebral Neurons from the Late Third Instar Larvae of Drosophila Melanogaster

作　　者：刘洋[1] 廖大清[1] 罗南富[1] 张贞雄[1] 刘进[1] 

机构地区：[1]四川大学华西医院麻醉与危重急救研究室,成都610041

出　　处：《四川大学学报（医学版）》2006年第6期938-942,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30070732);教育部博士点基金资助

摘　　要：目的探索果蝇晚三龄幼虫脑神经元急性分离的新方法,并对分离制备的脑细胞进行形态学观察和电生理学记录。方法解剖果蝇晚三龄幼虫脑组织,用胶原酶化学消化并辅以机械微振荡法分离制备单个细胞,用果蝇细胞培养液适当孵育。在倒置相差显微镜下对其进行形态学观察和分类,结合全细胞膜片钳技术对其电生理学特征进行初步研究。结果成功制备出三类大小和形态各异、活性较好的果蝇脑神经元,未见神经胶质细胞;以三型细胞为电生理学研究对象,记录到五种类型的全细胞外向钾电流。结论新建立的果蝇晚三龄幼虫脑细胞急性分离方法简便易行,稳定可靠。Objectives To develop a new method for acute isolation of the cerebral neurons from late third instar larvae of Drosophila melanogaster. Methods The dissociated cells were characterized by morphological observation and whole-cell voltage-clamp recording. The brains were dissected from late third instar larvae, torn into small fragments, and then were digested in the calcium-magnesium-free PBS solution endowed with collagenase for 45-60 minutes. Single cell was obtained by micro-shaking the digested fragments for 5 to 10 s until the clumps of the tissue were not invisible. Preparation of dispersed cells was incubated in the culture media of Drosophila for thirty minutes at room temperature （20± 1） ℃. Results All neurons studied were categorized into three types according to morphological observation： large （〉8 μm） round type Ⅰneuroblast-like cells （7%）, small （2-5μm） type Ⅰ cells （77%）, and intermediate-sized type E cells （16%）. Neurogliocytes were not found. The electrophysiological properties of three types of neurons were investigated by whole-cell voltage-clamp recording technique. Five types of outward potassium currents were detected readily. Conclusion Morphological and electrophysiological investigation showed that the method for acute isolation of Drosophila neurons is simple, available and stable.

关 键 词：果蝇 脑神经元 急性分离 形态学和电生理学 膜片钳 

分 类 号：Q421[生物学—神经生物学]