支配牙髓的痛感觉神经元的分离及其电生理特征研究

ISOLATION AND STUDY OF ELECTROPHYSIOLOGICAL CHARACTERISTICS OF NOCICEPTOR INNERVATING RAT DENTAL PULP

实验利用全细胞膜片钳技术研究了荧光染料核黄(NY)标记的支配牙髓的痛感觉神经元的电生理特征。将NY注入大鼠的牙髓,急性分离标记的三叉神经节(TG)神经元,即支配牙髓之痛敏神经元,测量NY标记细胞的直径,并和辣根过氧化物酶(HRP)标记的牙髓痛感觉神经元进行比较;同时检测NY标记细胞的膜电位,并进行全细胞膜片钳记录。结果显示:HRP标记细胞为中、小细胞,未见大细胞;而NY标记细胞均为小直径细胞,平均直径为24.25±3.10μm。膜片钳实验共检测了35个NY标记细胞,45.7%(16/35)的细胞对外加药物有反应,静息膜电位为45.48±7.55mV,其中25%(4/16)的细胞对外加ATP(10-4mol/L)有反应,为快去敏感内向电流;87.5%(14/16)的细胞对GABA(10-4mol/L)有反应,为慢去敏感内向电流;37.5%(6/16)的细胞对5-HT(10-4mol/L)有反应,均为快去敏感内向电流。结果提示:利用荧光示踪剂NY可以追踪、分离出支配牙髓的痛感觉神经元,其膜电位接近正常TG神经元的静息电位,且标记细胞对外加药物有反应。结果证明:NY不会影响细胞的生理活性,适用于全细胞膜片钳记录。利用该标记细胞可对痛感觉神经元膜上的受体及离子通道进行深入研究。

Whole-cell patch clamp technique was used to study the eleetmphysiological characteristics of nociceptive trigeminal ganglion （TG） neurons labeled by nuclear yellow （NY）. Horseradish peroxidase （HRP） and NY were injected into the dental pulp, respectively. The TG neurons labeled by HRP or NY, namely the nociceptors innervating the dental pulps, were freshly isolated to measure the diameter, respectively. In addition, membrane potential d neurons labeled by NY were measured and membrane-activated currents were recorded using whele-cell patch clamp. The results showed that HRP labeled neurons were small or medium sized cells, and NY labeled neurons were small sized cells （24.25 ±3.10 μm）, similar to HRP-positive neurons. Of 35 NY-positive neurons, 45.7% neurons （ 16/35 ） were responsive to drugs applied extracellularly, and the mean of membrane potential was -45.48 ±7.55 mY. Of 16 responsive neurons, 25 % cells （4/16 ） responded to ATP （ 10^ - 4 mol/L ） with an inward, mpidly-desonsitizing current ; 87.5 % cells （ 14/16 ） responded to GABA （ 10 ^-4 mol/L） with an inward, slowly-desensitizing current; and 37.5% cells （6/16） responded to 5-HT （ 10^ -4 mol/L） with an inward, rapidly-desensitizing current. It could be concluded that neurons labeled by NY can survive sufficiently and be isolated for studying the receptors and ion channels in the membrane using whole-cell patch clamp technique.