用于膜片钳研究的大鼠背根神经节细胞急性分离技术的改良

Modification of the procedure for dissociating rats' dorsal root ganglion cells used for patch clamp

目的通过技术改良,进一步摸索适于膜片钳实验的成年大鼠背根神经节(dorsal root ganglion,DRG)细胞的急性分离方法。方法应用显微外科技术获取成年大鼠DRG,采用改良的双酶顺序消化(先胶原酶,后胰蛋白酶)及双血清(胎牛血清+马血清)共同培养法,急性分离培养DRG。倒置显微镜下观察神经元的形态,选取已贴壁的细胞,用全细胞膜片钳技术记录其基本膜电生理特性。加入河豚毒素(tetrodotoxin,TTX)后再次记录DRG神经元的钠离子电流变化。结果本实验可获得形态良好、结构完整的单个DRG神经元,表面光亮,胞膜完整清晰,呈圆形或椭圆形。其静息膜电位为(55.22±4.39)mV,加入TTX后可记录到缓慢失活的河豚毒素不敏感(tetrodotoxin-resistant,TTX-r)的钠离子电流。其中,中等直径DRG神经元高阻封接成功率为73.2%,高于小直径和大直径DRG神经元的封接成功率(P<0.05)。结论经改良的DRG急性分离方法分离效率高、可操作性强。分离得到的中等直径DRG神经元状态良好,更适合膜片钳实验。

Objective To modify the previous procedure for dissociating rats dorsal root ganglion （DRG） cells used for whole-cell patch-clamp studies. Methods The rats DRGs were obtained by mierosurgical techniques, and then the DRG ceils were acutely dissociated and acquired with optimized culture method including dual enzyme digestion and dual serum incubation. After a short-time of culture, the DRG neurons were observed under inverted microscope and those with normal morphologic charactersties were selected for the further studies. The basic electrophysiological membrane character- istics and sodium channel currents were recorded by whole-cell patch-clamp technique before and after the administration of TI＇X, respectively. Results The cultured DRG cells, which were acutely dissociated and acquired with opti- mized method, were morphologically intact with a round or oval shape and smooth clear cell membrane. The rest- ing membrane potential was in a range of （55.22±4.39） mV. The slowly inactivated tetrodotoxin-resistant sodium currents were recorded after the administration of tetrodotoxin. The highest giga-seal successful rate （73.8%） with whole-cell patch-clamp was obtained in mid-diameter DRG neurons, which was significantly higher than that obtained from the small and large diameter neurons （P 〈 0.05）. Conclusions The optimized simple protocol, including dual enzyme digestion and dual serum incubation, can acquire morphologically intact rats DRG neurons with a high separation efficiency. The disso- ciated mid-diameter DRG neurons under these proedures are suitable for patch clamp research.