虎纹捕鸟蛛神经细胞急性分离培养及其电压门控通道膜片钳

Isolation and Culture of Nerve Cells from Ornithoctonus huwena and the Patch-clamp Study on the Voltage-gated Ion Channels in the Cultured Neurons

探索了虎纹捕鸟蛛(Ornithoctonus huwena)食道下神经细胞急性分离培养条件,并利用全细胞膜片钳技术对虎纹捕鸟蛛食道下神经细胞电压门控性钠、钾和钙通道的基本电生理学特性进行了研究。适合虎纹捕鸟蛛神经细胞离体培养的培养基为(g/L):葡萄糖0.7,果糖0.4,琥珀酸0.06,咪唑0.06,L-1513.7,Hepes 2.38,酵母粉2.8,乳白蛋白2.5,青霉素200 IU/ml,链霉素200 mg/ml,小牛血清15%;pH6.8。该培养基非常适合虎纹捕鸟蛛神经节神经细胞离体培养,细胞在温度(27±2)℃的培养箱中培养2～4h,培养的细胞数目多、结构完整、贴壁效果好,细胞近似汤勺形,有一个长的单极突起,大部分细胞在10～30μm之间。全细胞模式下可以记录到钠、钾和钙三种电压门控离子通道电流。钙电流为高电压激活电流,该电流能够被NiCl2完全抑制;钾电流为瞬时钾电流和延迟整流钾电流,这两类钾电流分别被细胞外液中的4-氨基吡啶和氯化四乙胺所阻断;钠电流为TTX敏感型电流。

In this article, the dissociation and culture of neurons isolated from the subesophageal ganglion （SUB） of the Ornithoctonus huwena are described. The basic electrophysiologieal properties of voltage-gated Na^＋ , K^＋ and Ca^2＋ channels on the cultured neurons were studied by means of whole-cell patch-clamp technique. The culture medium used for the nerve cells contained （g/L） ： glucose 0.7, fructose 0.4, sueeinic acid 0.06, imidazole 0.06, L- 15 13.7, Hepes 2.38, yeast extract 2.8, laetalbumin 2.5, penicillin 200 IU/ml, streptomycin 200 mg/ml, bovin calf serum 15 % ; pH 6.8. The suitable culture was 27 ± 2℃ for 2 - 4 h. Most ceils were in good condition and above 90% cells survived in the cell culture dishes. The shape of the soma of the nerve cell was in an ellipse and that of neural cell appreared like a spoon, with a single axon. The size of these cells varied from 10 to 30 μm. Whole-cell patch-clamp showed high-voltage-activated （HVA） calcium currents and two types of outward potassium currents including delayed rectifier potassium currents and rapid outward potassium currents on spider neurons. The potassium currents could be inhibited by TEA-CI and 4-AP. Sometimes, small voltage-gated sodium currents were also recorded in the experiment.