Nav1.8介导BmK I诱导痛觉传入的细胞与分子机制研究

发布时间：2018-05-28 04:13

本文选题：疼痛 + 电压门控钠通道　；参考：《上海大学》2015年博士论文

【摘要】：Bm K I,一个特异性电压门控钠通道位点3调制剂,已被验证能够诱导大鼠伤害性感受行为。Nav1.8在外周炎症痛以及神经痛发生、发展和维持过程中的参与角色日益受到研究者关注。那么,Nav1.8是否/又如何参与Bm K I诱导的疼痛发生、发展以及维持?对此,本文采用动物行为学及电生理学等手段,研究了Bm K I诱导大鼠疼痛模型中Nav1.8参与调控外周信号的细胞与分子机制。1.本研究发现,经Bm K I足底注射方式,两小时后的电生理记录发现,急性分离的大鼠DRG神经元中Nav1.8的电流密度显著上升,同时,其稳态激活和稳态失活曲线均向超极化方向偏移。鞘内和足底预处理A-803467(Nav1.8选择性阻断剂),显著地降低了大鼠自发痛和机械痛敏,但对热痛敏没有影响。结果清晰地提示,Bm K I诱导大鼠疼痛模型中,1)Nav1.8扮演了“首挡其冲”的应激者,即在2小时短时程内的应激反应触发了外周伤害性感受神经元的快速激活;2)Nav1.8是自发痛和机械痛的主要参与贡献者,即Nav1.8表达量的应激提升,通道蛋白门控动力学性质改变,增加了与自发痛和机械痛关联的神经元超兴奋性。2.经Bm K I在离体DRG神经元直接给药方式的电生理记录,Bm K I能够剂量依赖性地增大Nav1.8瞬时钠电流和持续性钠电流,并使Nav1.8稳态失活、和其快、慢失活向超极化方向偏移,显著减小了慢失活的电压依赖性,易化了通道的激活。结果表明,Bm K I直接调制了Nav1.8的门控动力学参数,快速地点燃了外周伤害性感受神经元的超兴奋性。3.分别合成了Nav1.8 DIV S3-S4之间,由22个残基组成(SIGSLLFSAILKSLENYFSPTL)的胞外环肽段和Nav1.5上相应区域由20个残基组成的(SIVGTVLSDIIQK—YFFSPTL)胞外环肽段(该肽段被认为是位点3的结合区域之一)。经表面等离子共振观察到,合成的Nav1.8胞外环多肽仍能与Bm K?结合,且结合速率比与合成的Nav1.5胞外环多肽的更高,解离速率更低,结合总量则相对较低。结果表明,Nav1.8胞外环四个插入氨基酸(SLEN)并未使Nav1.8失去对Bm K I的敏感性。由此提示,Bm K I与Nav1.8的分子结合机制有别于其他位点3毒素。
[Abstract]:Bm K I, a specific voltage-gated sodium channel site-3, has been shown to be able to induce nociceptive behavior in rats. Nav1.8 has attracted increasing attention in its role in the development, development and maintenance of peripheral inflammatory pain and neuralgia. So is / how does Nav1.8 participate in Bm K I induced pain generation, development, and maintenance? In this paper, the cellular and molecular mechanisms of Nav1.8 involved in the regulation of peripheral signals in Bm K I induced pain model in rats were studied by means of animal behavior and electrophysiology. In this study, the electrophysiological records of Bm K I plantar injection showed that the current density of Nav1.8 in DRG neurons was significantly increased after two hours, and at the same time, the current density of Nav1.8 was significantly increased in the acutely isolated rat DRG neurons. Both the steady-state activation and steady-state inactivation curves are shifted towards hyperpolarization. Intrathecal and plantar preconditioning with A-803467(Nav1.8 selective blocker significantly reduced spontaneous and mechanical pain sensitivity in rats but had no effect on thermal pain sensitivity. The results clearly indicated that in the pain model of rats induced by BmK I, 1 / Nav1.8 acted as a stressor. That is to say, the stress response during the 2 hour short time course triggered the rapid activation of peripheral nociceptive neurons. Naval 1.8 was the main contributor to spontaneous pain and mechanical pain, that is, the stress increase of Nav1.8 expression and the change of channel protein gated dynamics. Increased hyperexcitability of neurons associated with spontaneous and mechanical pain. 2. BmK I, an electrophysiological record of direct administration of Bm K I via DRG neurons in vitro, can increase the transient sodium current and persistent sodium current of Nav1.8 in a dose-dependent manner, and make Nav1.8 inactivate stably, and shift to hyperpolarization direction with fast and slow inactivation. The voltage dependence of slow inactivation was significantly reduced, and the activation of channels was facilitated. The results show that BmK I directly modulates the gated kinetic parameters of Nav1.8 and ignites the superexcitability of peripheral nociceptive neurons. The extracellular cyclic peptide of SIGSLLFSAILKSLENYFSPTL and the extracellular cyclic peptide of SIGSLLFSAILKSLENYFSPTL, composed of 20 residues, were synthesized between Nav1.8 DIV S3-S4 and SIGSLLFSAILKLENYFSPTL, respectively. The extracyclic peptide of SIGSLLFSAILKSLENYFSPTL is considered to be one of the binding regions of site 3. It was observed by surface plasmon resonance that the synthesized extracellular polypeptide of Nav1.8 could still be associated with Bm K? The binding rate was higher, the dissociation rate was lower and the total binding rate was lower than that with the synthesized Nav1.5 extracyclic polypeptides. The results showed that Nav1.8 did not lose its sensitivity to Bm K I. It is suggested that the molecular binding mechanism of BmK I with Nav1.8 is different from that of other site 3 toxin.
【学位授予单位】：上海大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R402

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