双孔钾离子通道TREK-1对氧糖剥夺星形胶质细胞和神经元功能的调节
发布时间：2018-08-06 17:06
【摘要】：研究目的：观察缺氧缺糖培养条件下,双孔钾离子通道TREK-1对大鼠皮质星形胶质细胞(Ast)存活率及谷氨酸(Glu)摄取功能的影响；Ast的功能状态与神经元之间的相互作用及TREK-1通道在其中的作用,探讨TREK-1通道是否能成为全新的治疗脑缺血疾病的潜在靶点。 研究内容：建立氧糖剥夺(oxygen and glucose deprivation, OGD)损伤模型,模拟体外脑缺血环境,检测Ast的存活率及Glu摄取功能,同时,给予花生四烯酸(AA)或甲硫氨酸(Met)改变TREK-1通道的功能,观察Ast存活率的变化,分析在体外OGD损伤下,Ast TREK-1通道的变化与Glu摄取功能变化间的关系。建立Ast与神经元细胞共培养体系,观察体外缺氧缺糖培养条件下,Ast和神经元TREK-1通道表达的变化,神经元存活率的变化,Ast和神经元谷氨酸受体亚型NR2B和mGluR、谷氨酸转运体GLT-1、凋亡通路中ERK和caspase-3的变化。给予AA或Met调节TREK-1通道的功能,观察以上指标的变化,以明确体外缺氧缺糖条件下,Ast功能状态对神经元的影响以及TREK-1通道在其中所起的作用。 研究结果：通过MTT检测,在生理条件下AA (5μmol/L.10μmol/L.20μmol/L、40μmol/L)和Met (1mmol/L、3mmol/L、10mmol/L、30mmol/L)对Ast的存活率均无影响,而缺氧缺糖条件下,AA和Met均可增加Ast的存活率。体外缺氧缺糖后,Ast对Glu的摄取出现代偿性增加,AA对OGD条件下Ast的Glu摄取有增强作用,而Met可以抑制OGD条件下Ast的Glu摄能力。通过乳酸脱氢酶(lactate dehydrogenase, LDH)释放检测,神经元-Ast共培养与神经元单独培养相比LDH释放量有所降低,给予Met后LDH释放量由47.84U/L下降到23.95U/L,说明Met可以减少神经元的死亡。通过RT-PCR检测显示,OGD条件下共培养神经元中TREK-1和GLT-1mRNA表达量下调,NR2B和mGluR mRNA表达量上升,共培养Ast中TREK-1和mGluR mRNA表达量下调,GLT-1和ERK mRNA表达量上升,给予AA后可下调TREK-1mRNA、NR2B mRNA及ERK mRNA的表达；给予Met后TREK-1mRNA及NR2B mRNA表达量均受到抑制,对GLT-1mRNA表达有双向调节作用。 研究结论：AA和Met对生理状态下Ast的存活率无显著影响,但能够改善OGD后Ast的存活率。AA与Met有助于增强OGD损伤后共培养神经元的存活,推测[REK-1通道功能的改变影响Ast与神经元之间的相互作用,但Met提高Ast的存活率与受体无明显联系,其促增殖原因有待进一步研究。
[Abstract]:Objective: to observe the effects of TREK-1 on the survival rate of (Ast) and the uptake of glutamate (Glu) in rat cortical astrocytes in hypoxic-glucose deficient culture. The functional state of Ast and the interaction between neurons and the role of TREK-1 channel in the interaction, to explore whether the TREK-1 channel can become a new potential target for the treatment of cerebral ischemic diseases. Content: establish oxygen glucose deprivation (oxygen and glucose deprivation, OGD) damage model, simulate the brain ischemic environment in vitro, detect the survival rate of Ast and Glu uptake function, at the same time, give arachidonic acid (AA) or methionine (Met) to change the function of TREK-1 channel. To observe the changes of Ast survival rate and to analyze the relationship between the changes of Ast TREK-1 channel and Glu uptake function under OGD injury in vitro. The co-culture system of Ast and neuronal cells was established to observe the expression of Ast and TREK-1 channels in cultured neurons under hypoxia and glucose deprivation in vitro. The changes of neuronal survival rate were related to the changes of neuronal glutamate receptor subtype NR2B and mGluR, glutamate transporter GLT-1, and ERK and caspase-3 in apoptotic pathway. AA or Met was given to regulate the function of TREK-1 channel, and the changes of the above indexes were observed to clarify the effect of Ast function on neurons under hypoxia and glucose deprivation in vitro and the role of TREK-1 channel in it. Results: under physiological conditions, AA (5 渭 mol/L.10 渭 mol/L.20 渭 mol / L ~ 40 渭 mol/L) and Met (1 mmol / L ~ (3) mmol / L ~ (10) mmol / L ~ (30 mmol / L) had no effect on the survival rate of Ast, but both AA and Met could increase the survival rate of Ast. After hypoxia and glucose deprivation in vitro, the uptake of Glu by Ast in vitro was increased. AA could enhance the Glu uptake of Ast under OGD, while Met could inhibit the Glu uptake of Ast under OGD condition. By detecting the release of lactate dehydrogenase (lactate dehydrogenase, LDH), the release of LDH in neuron Ast co-culture was lower than that in neuron culture alone, and the LDH release decreased from 47.84U/L to 23.95 U / L after Met administration, which indicated that Met could reduce the death of neurons. The expression of TREK-1 and GLT-1mRNA in co-cultured neurons was down-regulated by RT-PCR, and the expression of TREK-1 and mGluR mRNA in co-cultured Ast was down-regulated. The expression of TREK-1 and mGluR mRNA was down-regulated, and the expression of TREK-1mRNA-NR2B mRNA and ERK mRNA was down-regulated after treatment with AA. After Met, the expression of TREK-1mRNA and NR2B mRNA were inhibited, and the expression of GLT-1mRNA was regulated in both directions. Conclusion there is no significant effect on survival rate of Ast in physiological state by Met and% AA, but it can improve the survival rate of Ast after OGD. AA and Met can enhance the survival of co-cultured neurons after OGD injury. It is inferred that the changes of REK-1 channel function affect the interaction between Ast and neurons, but there is no obvious relationship between the increase of Ast survival rate and the receptor by Met, and the reasons for promoting proliferation need to be further studied.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
[Abstract]:Objective: to observe the effects of TREK-1 on the survival rate of (Ast) and the uptake of glutamate (Glu) in rat cortical astrocytes in hypoxic-glucose deficient culture. The functional state of Ast and the interaction between neurons and the role of TREK-1 channel in the interaction, to explore whether the TREK-1 channel can become a new potential target for the treatment of cerebral ischemic diseases. Content: establish oxygen glucose deprivation (oxygen and glucose deprivation, OGD) damage model, simulate the brain ischemic environment in vitro, detect the survival rate of Ast and Glu uptake function, at the same time, give arachidonic acid (AA) or methionine (Met) to change the function of TREK-1 channel. To observe the changes of Ast survival rate and to analyze the relationship between the changes of Ast TREK-1 channel and Glu uptake function under OGD injury in vitro. The co-culture system of Ast and neuronal cells was established to observe the expression of Ast and TREK-1 channels in cultured neurons under hypoxia and glucose deprivation in vitro. The changes of neuronal survival rate were related to the changes of neuronal glutamate receptor subtype NR2B and mGluR, glutamate transporter GLT-1, and ERK and caspase-3 in apoptotic pathway. AA or Met was given to regulate the function of TREK-1 channel, and the changes of the above indexes were observed to clarify the effect of Ast function on neurons under hypoxia and glucose deprivation in vitro and the role of TREK-1 channel in it. Results: under physiological conditions, AA (5 渭 mol/L.10 渭 mol/L.20 渭 mol / L ~ 40 渭 mol/L) and Met (1 mmol / L ~ (3) mmol / L ~ (10) mmol / L ~ (30 mmol / L) had no effect on the survival rate of Ast, but both AA and Met could increase the survival rate of Ast. After hypoxia and glucose deprivation in vitro, the uptake of Glu by Ast in vitro was increased. AA could enhance the Glu uptake of Ast under OGD, while Met could inhibit the Glu uptake of Ast under OGD condition. By detecting the release of lactate dehydrogenase (lactate dehydrogenase, LDH), the release of LDH in neuron Ast co-culture was lower than that in neuron culture alone, and the LDH release decreased from 47.84U/L to 23.95 U / L after Met administration, which indicated that Met could reduce the death of neurons. The expression of TREK-1 and GLT-1mRNA in co-cultured neurons was down-regulated by RT-PCR, and the expression of TREK-1 and mGluR mRNA in co-cultured Ast was down-regulated. The expression of TREK-1 and mGluR mRNA was down-regulated, and the expression of TREK-1mRNA-NR2B mRNA and ERK mRNA was down-regulated after treatment with AA. After Met, the expression of TREK-1mRNA and NR2B mRNA were inhibited, and the expression of GLT-1mRNA was regulated in both directions. Conclusion there is no significant effect on survival rate of Ast in physiological state by Met and% AA, but it can improve the survival rate of Ast after OGD. AA and Met can enhance the survival of co-cultured neurons after OGD injury. It is inferred that the changes of REK-1 channel function affect the interaction between Ast and neurons, but there is no obvious relationship between the increase of Ast survival rate and the receptor by Met, and the reasons for promoting proliferation need to be further studied.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
【参考文献】
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1 付振强;张博爱;贾延R，
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