摘要
目的:观察白细胞介素1β对河豚毒素敏感性钠电流的影响,拟从离子通道水平探讨白细胞介素1β调节颜面部痛的分子机制。方法:于2004-12/2005-11以2月龄左右的昆明种小鼠为实验对象,取双侧三叉神经节,将经分离得到三叉神经节细胞悬液经200目筛网过滤至直径35mm的培养皿内,待细胞贴壁后更换外液两次,应用EPC鄄9双通道膜片钳放大器行全细胞膜片钳记录。先经DAD给药系统向细胞喷射细胞外液5s,记录正常钠电流,再经DAD给药系统(ALA,USA)向细胞喷射实验用药,并记录给药后的钠通道电流。用细胞外液洗去残余药物,待钠电流恢复正常水平后,再向细胞喷射另一实验药物,进行下一个记录。两次记录之间的时间间隔不短于3min。药物效应观察完毕后,向细胞外喷射1μmol/L河豚毒素,根据被河豚毒素阻断程度判断所记录的钠电流类型。实验在室温22～25℃范围内进行。结果:①共稳定记录了81个三叉神经节细胞,其中在25.93%(21/81)的三叉神经节细胞上所记录的钠电流可被河豚毒素完全阻断,9.9%(9/81)的三叉神经节细胞上所记录的钠电流不能被河豚毒素阻断,大多数三叉神经节细胞(61.73%,50/81)上所记录的钠电流可部分被河豚毒素所阻断。②人重组白细胞介素1β显著抑制三叉神经节细胞河豚毒素敏感性INa,使INa的电流密度由给药前的(-86.92±8.08)PA/PF减为给药后的(-35.35±2.90)PA/PF(P<0.05);联合使用人重组白细胞介素1β及白细胞介素1ra也显著降低给药前INa的电流密度眼(-79.27±7.85),(-37.14±4.05)PA/PF,P<0.05演。③与给药前相比,人重组白细胞介素1β使河豚毒素敏感性INaⅠ～Ⅴ曲线显著上升,并使钠电流的最大激活电压向去极化方向偏移约10mV。④人重组白细胞介素1β使河豚毒素敏感性钠通道稳态激活曲线向去极化方向偏移熏V1/2给药前后去极化偏移差异有显著性意义眼(-40.03±2.75),(-35.35±3.47)mV,P<0.05演。⑤人重组白细胞介素1β不影响河豚毒素敏感性钠通道恢复时间常数,给药前后差异无显著性意义眼(4.55±0.58),(5.29±1.84)ms,P>0.05演。结论:白细胞介素1β直接作用于三叉神经节细胞河豚毒素敏感性钠通道,抑制其离子电流,可部分解释白细胞介素1β的颜面部镇痛作用。
AIM： To observe the effects of interleukin-1β （IL-1β） on tetrodotoxin-sensitive （TTX-S） sodium currents as well as probe into the molecular mechanism of IL-1β in regulation of tortua facies from the level of ion channel.
METHODS： Kunming mice of 2-month old were selected from December 2004 to November 2005. Mice were executed by cervical dislocation under anesthesia to obtain bilateral TGs. The TG cell suspension was filtered into a culture dish by 200 mesh, the diameter of which was 35 mm. The extracellular fluid was changed twice after cells adhered to the dish, and EPC-9 double-channel patch clamp amplifier was adopted to record the whole-cell patch clamp. The extracellular fluid was spraying on cells for 5 continuous seconds with DAD administration system, and normal sodium current was recorded, and then experimental drugs were sprayed on cells with DAD administration system （ALA, USA）, and the current of sodium channel after administration was recorded. The remaining drugs were washed off with extracellular fluid. Not until the sodium current returned to normal level, the next drug was sprayed on cells and the record was made. The time cell between each two records should be no less than 3 minutes. After the drug effect was observed, 1 μmol/L tetrodotoxin was sprayed on cells, and the type of recorded sodium current was judged according to the blocking degree of tetrodotoxin. The room temperature of the experiment was controlled in 22-25 ℃.
RESULTS： ① In this experiment, 81 TG cells were recorded, the recorded sodium current in 25.93 % （21/81） of which could be completely blocked by tetrodotoxin, and that in 9.9% （9/81） of TG cells could not be blocked by tetrodotoxin, while most of the recorded sodium current in TG cells （61.73%, 50/81） could be partially blocked by tetrodotoxin. ② The recombinant human IL-1β （rhIL-1β） significantly inhibited INa, the TTX-S of TG cells, and the current density of INa decreased from （-86.92±8.08） PA/PF before administration to （-35.35±2.90） PA/PF（P 〈 0.05） after administration. Combined using of rhIL-1β and IL-1ra also obviously decreased the current density of INa [（-79.27±7.85）, （-37.14±4.05） PA/PF, P 〈 0.05]. ③ The Ⅰ - Ⅴ curve of TTX-S INa remarkably ascended under the effects of IL-1β than that before administration, and the offset of the greatest activating voltage of sodium current was 10 mV towards the direction of depolarization. ④ The steady-state activation curve of TTX-S shifted to the direction of depolarization under the effect of rhIL-1β. The differences in depolarization shift before and after administration of Ⅴ 1/2 were marked [（-40.03±2.75）, （-35.35±3.47） mV, P 〈 0.05]. ⑤ The rhIL-1β did not affect the recovery-time constant of TTX-S sodium current, and there were no significant differences before and after the administration [（4.55±0.58）, （5.29±1.84） ms, P 〉 0.05].
CONCLUSION： IL-1β directly acts on the TTX-S sodium channels of TG cells and inhibits the ion current, which can partially explain the analgesic effects on facial area.
出处
《中国临床康复》
CSCD
北大核心
2006年第46期7-9,共3页
Chinese Journal of Clinical Rehabilitation
