胆囊收缩素CCK-8对小鼠背根神经元A-型钾通道电流作用及机制研究

发布时间：2018-12-25 13:01

【摘要】：目的:研究胆囊收缩素CCK-8(cholecystokinin-8;CCK-8)对小鼠背根神经节(dorsal root ganglion,DRG)神经元A-型钾通道电流及兴奋性作用和机制。方法:急性分离小鼠背根神经节(DRG)神经元,利用分子生物学方法鉴定其膜上CCK2受体的表达。利用全细胞膜片钳技术,研究CCK-8对小直径DRG神经元A-型钾通道电流及兴奋性的作用,并应用药理学方法研究其信号通路作用机制,最后在行为学的角度验证CCK-8的致痛作用。结果:本研究发现:1)在蛋白水平上,利用western blot和免疫荧光技术,小鼠背根神经节上有CCK2受体表达;2)电生理结果显示,CCK-8对DRG神经元上的A-型钾通道电流的抑制作用具有剂量依赖关系。100 nM CCK-8对A-型钾通道电流的抑制率约为25.9%;3)CCK-8对A-型钾通道电流的抑制作用能够被LY225910(CCK2R特异性阻断剂)、GDP-β-S(特异性G蛋白活性抑制剂)、PTX(百日咳毒素)所阻断。磷脂酰肌醇激酶(PI3K)的选择性阻断剂LY294002预先孵育背根神经节细胞后,可以阻断CCK-8对A-型钾通道的抑制作用,而孵育蛋白激酶A(PKA)的选择性阻断剂PKI6-22、磷脂酶C(PLC)选择性阻断剂U73122和蛋白激酶C(PKC)的选择性阻断剂GF109203X、Chelerythrine chloride、RO31-8220,则无此效果。4)CCK-8显著增加小直径背根神经节细胞的放电频率,此作用可被4-AP阻断。结论:CCK-8是通过作用于小鼠小直径背根神经节神经元上的CCK2R来抑制A-型钾通道的;其对A-型钾通道电流的抑制作用是通过PTX敏感的磷脂酰肌醇激酶(PI3K)启动的信号通路;CCK-8可增加背根神经节神经元细胞膜的兴奋性,进而参与疼痛调节。
[Abstract]:Aim: to study the effect and mechanism of cholecystokinin CCK-8 (cholecystokinin-8;CCK-8) on A-type potassium channel current and excitability of (dorsal root ganglion,DRG neurons in mouse dorsal root ganglion (DRG). Methods: (DRG) neurons were isolated from mouse dorsal root ganglion (DRG) and the expression of CCK2 receptor on the membrane was identified by molecular biology. The effects of CCK-8 on the current and excitability of A- type potassium channels in small diameter DRG neurons were studied by whole-cell patch clamp technique, and the mechanism of its signaling pathway was studied by pharmacological method. Finally, the pain effect of CCK-8 was verified from the behavioral point of view. Results: 1) at the protein level, CCK2 receptors were expressed in the dorsal root ganglion of mice using western blot and immunofluorescence techniques. 2) the electrophysiological results showed that the inhibitory effect of CCK-8 on the current of A- type potassium channel in DRG neurons was dose-dependent, and the inhibitory rate of 100 nM CCK-8 on the current of A- type potassium channel was about 25.9%. 3) the inhibitory effect of CCK-8 on A-type potassium channel current was blocked by LY225910 (CCK2R specific blocker) and GDP- 尾 -S (specific G protein activity inhibitor), PTX (pertussis toxin). LY294002, a selective inhibitor of phosphatidylinositol kinase (PI3K), preincubated dorsal root ganglion cells (DRG), which blocked the inhibitory effect of CCK-8 on the A- type potassium channel, while PKI6-22, a selective inhibitor of protein kinase A (PKA), was incubated. Phospholipase C (PLC) selective blocker U73122 and protein kinase C (PKC) selective blocker GF109203X,Chelerythrine chloride,RO31-8220, had no such effect. 4) CCK-8 significantly increased the discharge frequency of small diameter dorsal root ganglion cells. This effect can be blocked by 4-AP. Conclusion: CCK-8 inhibits A-type potassium channels by acting on CCK2R in small diameter dorsal root ganglion neurons of mice. The inhibitory effect of A- type potassium channel current was initiated by PTX sensitive phosphatidylinositol kinase (PI3K), and CCK-8 increased the excitability of dorsal root ganglion cell membrane and involved in pain regulation.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R402

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