【摘要】：目的:本研究旨在了解無鎂細(xì)胞外液誘導(dǎo)的海馬細(xì)胞癲癇模型中總dynamin 1及磷酸化dynamin 1的表達(dá)以及活動(dòng)依賴的批量內(nèi)吞作用(ADBE)的活動(dòng)水平,在細(xì)胞層面運(yùn)用dynamin 1的抑制劑和激動(dòng)劑觀察其對癲癇細(xì)胞的總dynamin 1和磷酸化dynamin 1以及ADBE的影響。方法:用無鎂細(xì)胞外液構(gòu)建海馬細(xì)胞癲癇模型,用Western blot技術(shù)檢測癲癇細(xì)胞、正常細(xì)胞以及經(jīng)dynamin 1的抑制劑(dynasore)和激動(dòng)劑(PIP2)處理過的癲癇細(xì)胞的總dynamin 1以及磷酸化dynamin1水平;并用Q-PCR技術(shù)檢測上述各細(xì)胞的dynamin 1的m RNA水平。運(yùn)用膜片鉗技術(shù)記錄癲癇細(xì)胞、正常細(xì)胞以及經(jīng)dynasore處理過的癲癇細(xì)胞的動(dòng)作電位情況。運(yùn)用活細(xì)胞熒光檢測技術(shù)觀察癲癇細(xì)胞、正常細(xì)胞對dextran的內(nèi)吞情況以及dynasore和PIP2對其內(nèi)吞的影響。結(jié)果:與正常細(xì)胞相比,癲癇細(xì)胞動(dòng)作電位頻率明顯升高,造模成功;癲癇細(xì)胞中去磷酸化dynamin 1水平明顯升高,對dextran的內(nèi)吞增強(qiáng);運(yùn)用dynasore處理癲癇細(xì)胞后,去磷酸化dynamin 1水平降低,對dextran的內(nèi)吞減少以及細(xì)胞動(dòng)作電位頻率下降。PIP2升高了癲癇細(xì)胞中去磷酸化dynamin 1水平,不改變細(xì)胞對dextran的內(nèi)吞。結(jié)論:我們的研究發(fā)現(xiàn),造模后癲癇細(xì)胞的興奮性明顯升高,癲癇細(xì)胞中去磷酸化的dynamin 1水平升高,其介導(dǎo)的ADBE途徑也增強(qiáng);運(yùn)用dynasore之后,癲癇細(xì)胞的dynamin 1去磷酸化水平降低,與之相對應(yīng)的ADBE途徑也減弱,同時(shí)降低了癲癇細(xì)胞的動(dòng)作電位頻率;我們推測dynasore可抑制無鎂外液誘導(dǎo)的癲癇細(xì)胞的興奮性,這個(gè)抑制作用可能是通過減弱ADBE途徑,從而抑制了神經(jīng)遞質(zhì)有效傳遞而實(shí)現(xiàn)的。
[Abstract]:Objective: to investigate the expression of total dynamin 1 and phosphorylated dynamin 1 in hippocampal cells induced by magnesium free extracellular fluid and the activity level of activity dependent mass endocytosis (ADBE). The effects of dynamin 1 inhibitor and agonist on total dynamin 1, phosphorylated dynamin 1 and ADBE in epileptic cells were observed at the cellular level. Methods: hippocampal epileptic model was established by extracellular solution without magnesium. The total dynamin 1 and phosphorylated dynamin1 levels in epileptic cells, normal cells and epileptic cells treated with dynamin 1 inhibitor (dynasore) and agonist (PIP2) were detected by Western blot technique. The m RNA level of dynamin 1 was detected by Q-PCR technique. The action potentials of epileptic cells, normal cells and epileptic cells treated with dynasore were recorded by patch clamp technique. The endocytosis of dextran by normal cells and the effects of dynasore and PIP2 on the endocytosis of epileptic cells were observed by living cell fluorescence assay. Results: compared with normal cells, the frequency of action potential of epileptic cells was significantly increased, and the model was successfully established. The level of dephosphorylated dynamin 1 in epileptic cells was significantly increased, and the endocytosis of dextran was enhanced. After dynasore was used to treat epileptic cells, The level of dephosphorylated dynamin 1 decreased, the endocytosis of dextran decreased and the frequency of action potential decreased. PIP2 increased the level of dephosphorylated dynamin 1 in epileptic cells, but did not change the endocytosis of dextran. Conclusion: our study found that the excitability of epileptic cells was significantly increased, the level of dephosphorylated dynamin 1 in epileptic cells was increased and the ADBE pathway mediated by dephosphorylation of dynamin 1 was also increased after the use of dynasore, and the dephosphorylation of dynamin 1 in epileptic cells was decreased after dynasore was used. The corresponding ADBE pathway was also weakened, and the action potential frequency of epileptic cells was decreased. We speculate that dynasore can inhibit the excitability of epileptic cells induced by magnesium free solution, which may be by weakening the ADBE pathway. Thus inhibiting the effective transmission of neurotransmitters.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R742.1

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本文編號：2278793