果蝇中枢神经元胰蛋白酶急性分离方法  

Acute isolation of cerebral neurons from the late third instar larvae of Drosophila melanogaster

作　　者：刘洋[1] 罗南富[1] 廖大清[1] 张贞雄[1] 李良文[2] 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 [2]四川省卫生管理干部学院解剖教研室,成都610041

出　　处：《四川生理科学杂志》2006年第1期1-4,共4页Sichuan Journal of Physiological Sciences

基　　金：国家自然科学基金资助(No.30070732)

摘　　要：目的:探索果蝇脑神经元胰蛋白酶急性分离的新方法。方法:解剖果蝇晚三龄幼虫脑组织,用胰蛋白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性进行初步研究。结果:该方法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:我们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。Objective： To develope a new method of acute isolation of 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 techniques. Brains were dissected from late third instar larvae and tom them into small fragments that were incubated for 10 minutes in the calcium/magnesium-free PBS solution endowed with 0. 0125% trypsin. Single cell was obtained by micro-shaking the digested fragments for 5 to lOs until the clumps of the tissue were invisible. Preparation of dispersed cells was incubated in the culture media of Drosophila for thirty minutes at room temperature. Results： All neurons studied were categorized into three types according to morphological observation： large （ 〉 glum） round type Ⅰ neuroblast-hke cells（7% ）, small （2 -5μm） type Ⅱ cells（77% ）, and intermediate sized type m cells（ 16% ）. Neurogliocytes were not found. The electrophysiological properties of the three types of neurons were studied by whole-cell voltage-clamp recording techniques. Five types of outward potassium currents were detected readily. Conclusion： This study showed that the methods of acute isolation of Drosophila neurons are simple, available and stable.

关 键 词：果蝇 神经元 急性分离 膜片钳 

分 类 号：Q965-33[生物学—昆虫学]