Dynamin 1的磷酸化状态与神经元兴奋性的相关性研究
发布时间：2018-10-18 10:03
【摘要】：目的:本研究旨在了解无镁细胞外液诱导的海马细胞癫痫模型中总dynamin 1及磷酸化dynamin 1的表达以及活动依赖的批量内吞作用(ADBE)的活动水平,在细胞层面运用dynamin 1的抑制剂和激动剂观察其对癫痫细胞的总dynamin 1和磷酸化dynamin 1以及ADBE的影响。方法:用无镁细胞外液构建海马细胞癫痫模型,用Western blot技术检测癫痫细胞、正常细胞以及经dynamin 1的抑制剂(dynasore)和激动剂(PIP2)处理过的癫痫细胞的总dynamin 1以及磷酸化dynamin1水平;并用Q-PCR技术检测上述各细胞的dynamin 1的m RNA水平。运用膜片钳技术记录癫痫细胞、正常细胞以及经dynasore处理过的癫痫细胞的动作电位情况。运用活细胞荧光检测技术观察癫痫细胞、正常细胞对dextran的内吞情况以及dynasore和PIP2对其内吞的影响。结果:与正常细胞相比,癫痫细胞动作电位频率明显升高,造模成功;癫痫细胞中去磷酸化dynamin 1水平明显升高,对dextran的内吞增强;运用dynasore处理癫痫细胞后,去磷酸化dynamin 1水平降低,对dextran的内吞减少以及细胞动作电位频率下降。PIP2升高了癫痫细胞中去磷酸化dynamin 1水平,不改变细胞对dextran的内吞。结论:我们的研究发现,造模后癫痫细胞的兴奋性明显升高,癫痫细胞中去磷酸化的dynamin 1水平升高,其介导的ADBE途径也增强;运用dynasore之后,癫痫细胞的dynamin 1去磷酸化水平降低,与之相对应的ADBE途径也减弱,同时降低了癫痫细胞的动作电位频率;我们推测dynasore可抑制无镁外液诱导的癫痫细胞的兴奋性,这个抑制作用可能是通过减弱ADBE途径,从而抑制了神经递质有效传递而实现的。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.1
本文编号：2278793
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.1
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