δ-阿片受体在PTZ诱导癫痫中的作用

发布时间：2018-07-12 20:29

本文选题：癫痫 + δ-阿片受体　；参考：《大连理工大学》2014年硕士论文

【摘要】：本实验利用戊四氮(PTZ)建立体外癫痫模型,在细胞水平研究癫痫可能的发病机理,进一步研究δ-阿片受体(DOR)在PTZ诱导的体外癫痫模型中的作用,揭示DOR在癫痫的发生和控制方面所起的作用。本实验中用PTZ处理星形胶质细胞-神经元共培养体系细胞,建立癫痫模型。MTT实验表明,PTZ对细胞活性有一定的影响,膜片钳实验结果表明,PTZ能够明显的增高原代神经元IK电流峰值,呈一定的浓度依赖性和电压依赖性,能够使IK稳态激活曲线和稳态失活曲线显著地向右移动,增高半数激活电压和半数失活电压,减缓通道的激活过程和失活过程。根据这些实验数据,用lOmMPTZ处理细胞24h建立体外癫痫模型。在钾通道电流中,延迟整流型钾电流(IK)和内向整流钾电流(IKir)与癫痫的发生有关,所以在实验中应用膜片钳技术,研究PTZ对神经元Kv2.1电流和星形胶质细胞Kir4.1电流的影响。实验结果表明,lOmM PTZ能够明显的增大Kir4.1通道的电流值。lOmM PTZ能够增加Kv2.1电流幅度；增大通道的激活时间常数和失活时间常数来影响通道的门控特性；可以增大通道的半数激活电压和半数失活电压,减慢通道的激活和失活。最近的研究表明,DOR可能在癫痫的发生上有不可忽视的作用。在本实验中中利用DOR的特异性激动剂DADLE,研究DOR在癫痫模型中的作用。实验结果表明,1μM、5μM、10μMDADLE的预处理能够抑制lOmM PTZ诱导的模型细胞NO、Glu水平,腺苷酸脱氨酶(ADA)活性,线粒体膜电位的增加,即能够明显的抑制PTZ的诱导作用。根据这些实验结果,选择5μM DADLE来调节DOR的活性,用于后续试验。为了研究DOR与癫痫的异常放电之间的关系,我们研究了5μM DADLE对神经元Kv2.1通道和星形胶质细胞Kir4.1通道的影响。结果表明,5μM DADLE对Kir4.1电流有抑制作用。5μM DADLE能够显著地降低神经元Kv2.1电流峰值。同时5μM DADLE能够降低通道的激活时间常数和失活时间常数。同时,也能够使通道的激活曲线和失活曲线向电压的负方向移动,加快了通道的失活与激活。以上结果表明,10mM PTZ能够有效的诱导癫痫模型的建立,而用DADLE来激活DOR能够抑制由PTZ诱导的模型细胞活性和兴奋性的变化,在一定程度上抑制癫痫的发生。
[Abstract]:In this study, pentylenetetrazol (PTZ) was used to establish an in vitro epileptic model. The possible mechanism of epilepsy was studied at the cell level, and the role of 未 -opioid receptor (DOR) in PTZ induced epilepsy in vitro was further studied. To reveal the role of DOR in the occurrence and control of epilepsy. In this experiment, PTZ was used to treat astrocyte-neuron co-culture system cells, and the epilepsy model was established. MTT assay showed that PTZ had a certain effect on cell activity. The results of patch clamp experiment showed that PTZ could significantly increase the peak value of IK current in primary neurons in a concentration-and voltage-dependent manner, and could make the steady-state activation curve and steady-state inactivation curve of IK move significantly to the right. Increase half activation voltage and half inactivation voltage, slow down the activation and inactivation of channel. According to these experimental data, lOmMPTZ was used to treat the cells for 24 hours to establish an in vitro epileptic model. In potassium channel currents, delayed rectifier potassium currents (IK) and inward rectifier potassium currents (IKir) are associated with epilepsy, so the effects of PTZ on neuron Kv2.1 currents and astrocyte Kir4.1 currents were investigated by patch clamp technique. The experimental results show that the current value of Kir4.1 channel. LOmM PTZ can increase the amplitude of Kv2.1 current, increase the activation time constant and inactivation time constant to influence the gated characteristics of the channel. It can increase the half activation voltage and half inactivation voltage of the channel and slow down the activation and inactivation of the channel. Recent studies have shown that DOR may play an important role in the development of epilepsy. In this study, DOR was used as a specific agonist to study the role of DOR in epileptic model. The results showed that the pretreatment of 1 渭 M ~ 5 渭 M ~ 5 渭 M ~ + 10 渭 M DADLE could inhibit the level of Glu, the activity of adenylate deaminase (ADA) and the increase of mitochondrial membrane potential in model cells induced by lOmM PTZ, that is, it could obviously inhibit the induction of PTZ. According to these results, 5 渭 M DADLE was selected to regulate the activity of DOR. In order to study the relationship between DOR and abnormal discharges of epilepsy, we studied the effects of 5 渭 M DADLE on Kv2.1 channel and Kir4.1 channel of astrocytes. The results showed that 5 渭 M DADLE inhibited the Kir4.1 current significantly, and the peak value of Kv2.1 current was significantly decreased by 0.5 渭 M DADLE. At the same time, 5 渭 M DADLE can decrease the activation time constant and the inactivation time constant of the channel. At the same time, the channel activation curve and the inactivation curve can move to the negative direction of the voltage, which accelerates the channel inactivation and activation. The above results indicate that 10mm PTZ can effectively induce epilepsy model, and DOR activated by DADLE can inhibit the changes of cell activity and excitability induced by PTZ, and to some extent inhibit the occurrence of epilepsy.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

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