起搏通道电生理特性研究

发布时间：2018-04-20 09:49

本文选题：HCN通道 + KT5720　；参考：《华中科技大学》2013年博士论文

【摘要】：细胞是生命活动的基本单位。一切有机体都是由细胞构成的，它是有机体生长与发育的基础，也是遗传的基本单位，因此，没有细胞就没有完整的生命。所有的动物细胞都由一层薄膜所包围，这就是细胞膜(plasma membrane)，细胞膜的作用是保持细胞内物质成分的稳定。但是，，细胞膜并不是完全封闭的，细胞需要跟外界交换物质和能量，细胞膜上的离子通道就是具备这种功能的蛋白质，离子通道可以分为配体门控通道、电压门控通道、胞内信号门控通道和机械敏感通道四种类型。超极化激活的环核苷酸门控(hyperpolarization-activated cyclic nucleotide-gated，HCN)通道(起搏通道)是一种能被电压和环磷酸腺苷(cAMP)调控的、无选择性阳离子通道，它介导的电流是Ih。Ih广泛的参与了对一系列生理活动的调控，包括心脏和神经元节律，某些可兴奋细胞的静息电位，神经信号的传递以及信号在树突中的整合等。Ih受自主神经系统和神经内分泌系统的调节以及某些病理状态的影响，Ih异常会导致心脏及精神方面的多种疾病。因此，研究Ih有重要的生理学意义。本文着重应用电生理膜片钳、全细胞钙成像、神经疼痛模型等技术对HCN通道的调控机制、在神经疼痛中的作用及其药理学特性进行了研究和探讨。本文主要研究结果和结论如下：（1）PKA信号通路在调控HCN通道活性中的作用。PKA广泛分布于细胞体内，参与了对一些列重要生理活动的调控。然而，抑制PKA对HCN通道活性有何影响，目前仍不清楚。为了弄清这一问题，我们选择了PKA的选择性抑制剂KT5720。在新鲜分离的大鼠DRG神经元上，综合运用全细胞电压钳、电流钳、单通道记录以及钙成像技术。揭示了PKA信号通路在调控HCN通道活性中发挥了极为重要的作用，KT5720抑制HCN通道活性，使其电流密度减小，半数开放电压向超极化方向移动，并使其通道开放的时间常数增加。此外，KT5720抑制PKA使DRG神经元上的动作电位减小、胞内Ca2+浓度降低，从而抑制DRG神经元的兴奋性。（2）HCN通道调控神经疼痛。神经疼痛通常由神经损伤引发，因其具有发病率高、难治愈等特点，已成为当今医学界亟待解决的主要问题之一。另一方面，DRG神经元是感觉传入的第一级神经元，在疼痛信号的传导过程中发挥着不可或缺的重要作用。因此，我们选择DRG神经元为研究对象，利用大鼠幸免神经损伤模型。综合运用多种电生理膜片钳实验方法，研究和探讨了HCN通道在调控神经疼痛中的机制，揭示了HCN通道是触发和维持神经疼痛的关键因素。在幸免神经损伤诱导的疼痛状态下，DRG神经元上的HCN通道活性显著增加，异位放电频率变快，且DRG形态发生变化。因此，HCN通道是有效治疗神经疼痛的潜在靶点。（3）麻黄碱对HCN通道的影响。麻黄碱作为一种肾上腺受体激动药，具有加强心收缩力、增加心输出量、加快心率、升高血压等作用。为了弄清麻黄碱的这些作用是否是通过HCN通道来实现的以及它对HCN通道电流有什么影响。我们采用传统的全细胞膜片钳实验方法，在新生的大鼠海马神经元和HEK细胞上记录到HCN通道电流Ih。实验结果显示，麻黄碱对Ih有抑制作用。经分析，我们认为HCN通道可能具备某种反馈及保护功能，即当血压和心率上升时，HCN通道能部分阻滞这种上升趋势，从而保护机体健康和生命安全。
[Abstract]:Cell is the basic unit of life activity . All organism is composed of cells , it is the base of organism growth and development , it is the basic unit of inheritance , therefore , there is no cell without complete life . All animal cells are surrounded by a layer of membrane .

The hyperpolarization activated cyclic nucleotide - gated ( HCN ) channel is a non - selective cation channel which can be regulated by voltage and cyclic adenosine monophosphate ( cAMP ) . Its current is Ih . Ih is extensively involved in regulation of a series of physiological activities , including heart and neuronal rhythm , resting potential of certain excited cells , transmission of nerve signals , and integration of signals in the dendritic cells . Ih is influenced by autonomic nervous system and neuroendocrine system and the integration of signals in the dendritic cells . Ih can lead to various diseases in the heart and spirit . Therefore , it is important to study Ih .

This paper focuses on the mechanism of HCN channel regulation by using the techniques of electro - physiological patch clamp , whole - cell calcium imaging and neural pain model , and studies and discusses the role and pharmacology of HCN channel .

The main findings and conclusions are as follows :

( 1 ) In order to clarify this problem , we have selected selective inhibitor KT 5720 . In order to understand this problem , we have chosen selective inhibitor of protein kinase , KT 5720 . In order to understand this problem , we have chosen selective inhibitor of the protein of the activity of HCN channel . In order to understand this problem , we have chosen the selective inhibitor of the protein kinase , KT 5720 . In order to find out the problem , we have chosen the selective inhibitor of protein kinase , KT 5720 . In order to find out the problem , we have disclosed the activity of the channel in the freshly isolated rat DRG neurons , and the half - open voltage is decreased , and the time constant of the channel opening is increased . In addition , the inhibition of the activity of the pathway of the channel is reduced , and the concentration of Ca 2 + in the cells is decreased , so that the excitatory of the DRG neurons is inhibited .

In this paper , we select DRG neurons as the research objects and play an important role in the conduction of pain signals .

( 3 ) The effect of ephedrine on HCN channel was studied . The effect of ephedrine on HCN channel was studied .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.04

【参考文献】