本文選題：癲癇 + δ-阿片受體�。� 參考：《大連理工大學(xué)》2014年碩士論文

【摘要】：本實驗利用戊四氮(PTZ)建立體外癲癇模型,在細(xì)胞水平研究癲癇可能的發(fā)病機(jī)理,進(jìn)一步研究δ-阿片受體(DOR)在PTZ誘導(dǎo)的體外癲癇模型中的作用,揭示DOR在癲癇的發(fā)生和控制方面所起的作用。 本實驗中用PTZ處理星形膠質(zhì)細(xì)胞-神經(jīng)元共培養(yǎng)體系細(xì)胞,建立癲癇模型。MTT實驗表明,PTZ對細(xì)胞活性有一定的影響,膜片鉗實驗結(jié)果表明,PTZ能夠明顯的增高原代神經(jīng)元IK電流峰值,呈一定的濃度依賴性和電壓依賴性,能夠使IK穩(wěn)態(tài)激活曲線和穩(wěn)態(tài)失活曲線顯著地向右移動,增高半數(shù)激活電壓和半數(shù)失活電壓,減緩?fù)ǖ赖募せ钸^程和失活過程。根據(jù)這些實驗數(shù)據(jù),用lOmMPTZ處理細(xì)胞24h建立體外癲癇模型。 在鉀通道電流中,延遲整流型鉀電流(IK)和內(nèi)向整流鉀電流(IKir)與癲癇的發(fā)生有關(guān),所以在實驗中應(yīng)用膜片鉗技術(shù),研究PTZ對神經(jīng)元Kv2.1電流和星形膠質(zhì)細(xì)胞Kir4.1電流的影響。實驗結(jié)果表明,lOmM PTZ能夠明顯的增大Kir4.1通道的電流值。lOmM PTZ能夠增加Kv2.1電流幅度；增大通道的激活時間常數(shù)和失活時間常數(shù)來影響通道的門控特性；可以增大通道的半數(shù)激活電壓和半數(shù)失活電壓,減慢通道的激活和失活。 最近的研究表明,DOR可能在癲癇的發(fā)生上有不可忽視的作用。在本實驗中中利用DOR的特異性激動劑DADLE,研究DOR在癲癇模型中的作用。實驗結(jié)果表明,1μM、5μM、10μMDADLE的預(yù)處理能夠抑制lOmM PTZ誘導(dǎo)的模型細(xì)胞NO、Glu水平,腺苷酸脫氨酶(ADA)活性,線粒體膜電位的增加,即能夠明顯的抑制PTZ的誘導(dǎo)作用。根據(jù)這些實驗結(jié)果,選擇5μM DADLE來調(diào)節(jié)DOR的活性,用于后續(xù)試驗。 為了研究DOR與癲癇的異常放電之間的關(guān)系,我們研究了5μM DADLE對神經(jīng)元Kv2.1通道和星形膠質(zhì)細(xì)胞Kir4.1通道的影響。結(jié)果表明,5μM DADLE對Kir4.1電流有抑制作用。5μM DADLE能夠顯著地降低神經(jīng)元Kv2.1電流峰值。同時5μM DADLE能夠降低通道的激活時間常數(shù)和失活時間常數(shù)。同時,也能夠使通道的激活曲線和失活曲線向電壓的負(fù)方向移動,加快了通道的失活與激活。 以上結(jié)果表明,10mM PTZ能夠有效的誘導(dǎo)癲癇模型的建立,而用DADLE來激活DOR能夠抑制由PTZ誘導(dǎo)的模型細(xì)胞活性和興奮性的變化,在一定程度上抑制癲癇的發(fā)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.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R742.1

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本文編號：2118373