棉铃虫幼虫神经细胞的急性分离培养及其电压门控通道的膜片钳研究

Acute isolation and culture of nerve cell from the cotton bollworm and the patch-clamp study on the voltage-gated ion channels in the cultured neurons

探索了棉铃虫Helicoverpaarmigera幼虫神经细胞的急性分离与体外培养的条件 ,并利用全细胞膜片钳技术首次对棉铃虫幼虫急性分离神经细胞的电压门控性钠、钾和钙通道的基本电生理学特性进行了研究。结果表明 ,棉铃虫幼虫中枢神经细胞在TC 10 0、L 15和Grace培养基中均可贴壁生长 ,在DMEM培养基中基本不能存活。在TC 10 0培养基分别与其它三种培养基按一定比例混合形成的培养液中 ,TC 10 0与L 15等量混合培养液更适合于神经细胞的生长。全细胞电压钳条件下 ,可分别记录到电压门控性钠、钾和钙通道电流。钙电流特征为高电压激活、缓慢失活 ;钠电流对河豚毒素敏感 ;钾电流可被细胞外液中的氯化四乙胺和 4 氨基吡啶抑制。

The dissociation and culture of cells isolated from the ventral nerve cord of the larva of the cotton bollworm, Helicoverpa armigera are described. The propterties of the voltage-gated ion channels of the cultured neurons were studied with patch-clamp technique in the whole-cell configuration. Neurons survived and grew fast in either TC-100, Leibovitz's L-15 or Grace medium supplemented with glutathione, glutamine, glucose and 10% foetal calf serum. The medium made up of equal parts of TC-100 and Leibovitz's L-15 enabled better adherence, survival and growth. Most neurons showed neurite growth exhibiting unipolar, bipolar or multipolar. Neural cell bodies readily formed seals with patch pipettes, allowing stable, whole-cell patch-clamp recordings. Depolarization of the cell under voltage-clamp resulted in TTX-sensitive sodium currents, high-voltage activated calcium currents and two types of outward potassium currents. The potassium currents could be decreased by TEA-Cl and 4-AP.