【作者】 骆翔；

【导师】 王伟；

【作者基本信息】 华中科技大学 ， 神经病学， 2008， 博士

【摘要】 第一部分糖氧剥夺对大鼠海马星型胶质细胞膜电位的影响目的:观察大鼠海马星型胶质细胞对缺氧缺糖损伤的急性电生理反应。方法:采用出生后23天的大鼠建立体外大鼠急性海马脑片的缺氧缺糖模型(OGD模型),应用急性海马切片及膜片钳技术记录星型胶质细胞在正常生理条件下和OGD过程中膜电位的变化。结果:正常生理条件下,大鼠海马CA1区成熟的星型胶质细胞呈现低膜电阻(Rm)并且其膜电位呈线性电导,可被一系列的去极化或者超极化电压诱导,星型胶质细胞的静息膜电位由细胞内外钾离子梯度决定。持续灌注OGD溶液30min时,星型胶质细胞膜电位显示多相性去极化,而不是线性去极化。表现为OGD开始至11min的一个小幅度的去极化阶段,此期结束时,膜电位平均值在3.8±1.6mV;紧接着是6min的加速去极化,在17min达到稳定的高峰并且出现幅度6.0±1.8mV的去极化;然后为第二个平台期。OGD后,去极化的膜电位很快恢复到静息电位水平,并会跟随一个短暂的比OGD处理前水平低的超极化3.4±1.6mV。非持续灌流OGD组星型胶质细胞膜电位反应趋势与持续灌注OGD组相似,但其膜电位均值显示更强的去极化(P＜0.05)。结论:大鼠海马CA1区成熟的星型胶质细胞表达特征性的具有线性I-V关系的被动电传导,并且具有低的膜电位及膜电阻。成熟胶质细胞的膜电位接近钾的平衡电位,表明钾通道参与了其电传导;星型胶质细胞对OGD损伤比神经元具有更强的耐受性,OGD损伤可诱导海马星型胶质细胞膜电位产生多相性去极化,有其独特的电生理反应模式。第二部分糖氧剥夺导致大鼠海马星型胶质细胞膜电位反应的机制探讨目的:探讨有关星型胶质细胞对氧糖缺失损伤的急性电生理反应的基本机制。方法:应用急性海马切片及膜片钳技术记录星型胶质细胞在OGD过程中膜电位的变化以及各种抑制剂干预对其膜电位的变化影响。建立体外大鼠急性海马脑片的缺氧缺糖模型(OGD模型),采用出生后23天的大鼠,分为OGD组、OGD+药物干预组,OGD溶液中加入相应受体的抑制剂:AMPA受体抑制剂(30 mmol/L NBQX)、NMDA受体抑制剂D,L-AP5(50 mmol/L)、谷氨酸转运体受体抑制剂TBOA(100 mmol/L)、P2X受体非选择性受体阻滞剂suramins(100 mmol/L)、GABAA受体的阻滞剂bicuculline(10mmol/L)以及缝隙连接阻滞剂MFA(100mmol/L),以观察星型胶质细胞的缝隙连接、离子受体和转运体的激活对其膜电位变化的影响;OGD溶液中加入iodoacetate(2mmol/L),或antimycin(25 mmol/L)和rotenone(50 mmol/L)来检测糖酵解和线粒体氧化磷酸化对星型胶质细胞膜电位变化作用;结果:星型胶质细胞的缝隙连接、离子受体和转运体的抑制剂没有影响OGD诱导的星型胶质细胞去极化膜电位(P＞0.05);离子受体和转运体的抑制剂联合应用也没有对星型胶质细胞去极化膜电位产生显著影响(P＞0.05);当糖酵解和氧化磷酸化途径被抑制时OGD诱导星型胶质细胞膜电位产生快的去极化。糖酵解抑制剂iodoacetate干预时星型胶质细胞在OGD8分钟时产生一个iodoacetate与antimycin联合应用时相似的膜电位变化。结论:OGD诱导的特征性的星型胶质细胞膜电位变化不受星型胶质细胞上的谷氨酸、GABA受体以及转运体激活的影响,说明细胞外钾的变化导致了OGD后星型胶质细胞膜电位变化;糖酵解途径是星型胶质细胞在OGD早期维持细胞膜上钠钾泵功能及保持膜电位的主要能量来源。第三部分缺血缺氧对体外培养星型胶质细胞细胞周期和周期相关蛋白的影响目的:观察缺血缺氧损伤对星型胶质细胞细胞周期及细胞周期相关蛋白的影响。方法:用流式细胞仪及Brdu掺入法检测缺血缺氧后不同时间点星型胶质细胞细胞周期变化和细胞的增殖活力;用荧光免疫细胞化学技术测定胶质细胞纤维酸性蛋白(GFAP)、增殖细胞核抗原(PCNA)及细胞周期蛋白cyclinD1的表达水平结果:体外缺血缺氧损伤后星型胶质细胞S期较正常组明显增高,6小时达高峰,Brdu掺入法显示损伤后6小时星型胶质细胞的增殖活力最高,而随后S期细胞数目及细胞增殖活力都呈下降趋势。在缺血缺氧早期,GFAP阳性染色增强,6小时最高;缺血缺氧12h后GFAP阳性染色变弱,PCNA阳性反应损伤后表达增加,6h表达最高,而cyclinD1的表达在损伤后逐渐增加,在24h时达高峰。结论:缺血缺氧损伤激活星型胶质细胞,使其进入新的细胞周期,出现细胞的增殖反应;PCNA以及cyclinD1参与了损伤后星型胶质细胞的修复和增殖;细胞周期事件与星型胶质细胞的增殖活化密切相关。更多还原

【Abstract】 Part One The effections of Oxygen and Glucose Deprivation on the Electrophysiological Characters of Astrocytes in Rat HippocampusObjective:To study the effections of Oxygen and Glucose Deprivation on the electrophysiological characters of astrocytes in rat hippocampus.Methods:In this study,acute hippocampal slices were prepared from 23-day-old rats. we applied glucose and oxygen-deficient artificial cerebrospinal fluid(aCSF)(oxygen and glucose deprivation(OGD) solution) to acute rat hippocampus slices and examined astrocyte Vm in the physiological condition and OGD condition by patch-clamp recording.Result:In the physiological condition,mature astrocytes predominantly express linearⅠtoⅤconductance that can be evoked by a series of de-and hyperpolarization voltage steps in CA1 area.Such a linear whole-cellⅠtoⅤrelationship is predominately due to the expression of leak K+ channels,When the recording chamber was continually perfused with OGD solution for 30 mins,the astrocyte Vm showed a multiphasic,rather than linear,depolarization.This was characterized by an initial small-amplitude depolarization phase in the first 11min of OGD process.At the end of this phase,the Vm depolarization amounced to an average o(?)mV; followed by a 6min accelerating depolarizing period,and a second plateau.On OGD withdrawal,this depolarized Vm returned rapidly to the resting Vm level and was followed by a transient further 3.4±1.6mV hyperpolarization below the initial pre-OGD level.The mean astrocyte Vm response over 30 mins OGD under this nonperfusion condition showed similar response and a much stronger depolarization(P＜0.05).Conclusion:Mature astrocytes predominantly express linearⅠtoⅤconductance in CA1 area,and they have a low membrane potential and membrane resistance.The reversal potential of mature astrocytes is much closer to the equilibrium potential of K. This indicated K+ channels were involed in astrocytes conductance;Astrocytes are more resistant to OGD damage than neurons;OGD Induces a multiphase astrocyte membrane potential depolarization, Part Two The Underling mechanisms of the Electrophysiological Changes of Rat Hippocampal Astrocytes following Oxygen and Glucose DeprivationObjective:To investigate the basical mechanisms of the acute electrophysiological response of astrocytes in rat hippocampus following OGD.Methods:In this study,acute hippocampal slices were prepared from 23-day-old rats. we applied glucose and oxygen-deficient artificial cerebrospinal fluid(aCSF)(oxygen and glucose deprivation(OGD) solution) to acute rat hippocampus slices and examined astrocyte Vm in OGD condition and inhibitors interfered condition by patch-clamp recording.We added inhibitors for AMPA receptors(30 mmol/L NBQX), NMDA receptors(50 mmol/L D,L-AP5),glutamate transporters(100 mmol/L TBOA)、P2X receptors antagonist(100 mmol/L suramins)、GABAA receptor antagonist(10 mmol/L bicuculline) and gap junction blocker(100mmol/L MFA) to the OGD solution, then we observed the effections of astrocytic gap junction、ionotropic receptors and transporter on OGD induced astrocyte membrane potential changes;we added iodoacetate(2 mmol/L)、antimycin(25 mmol/L)and rotenone(50 mmol/L) to the OGD solution to investigated the effections of glycolytic and mitochondrial oxidative phosphorylation on OGD induced astrocyte membrane potential changes.Result:These inhibitors did not significantly affect OGD-induced astrocyte Vm depolarization(P＞0.05);When glycolytic and mitochondrial ATP production was inhibited,astrocytes showed a rapid depolarization,We found that the glycolysis inhibitor iodoacetate(2 mmol/L) produced a similar Vm changes as the three inhibitors applied together for an 8-min OGD treatment.Conclusion:OGD induced astrocyte membrane potential changes were not effected by the gap junction、glutamate receptors/transporters and GABA receptors,this indicated extracellular K+ increase was primarily responsible for the astrocytes embrane potential multiphasic.changes following OGD;Glycolysis plays a preferential role in keeping the Vm and Na-K-ATP pump functions in the early period of OGD. Part Three Effects of ischemia and anoxia on cell activation and cell cycle of cultured astrocyte in vitro.Objective:To study the effects of ischemia and anoxia on cell activation and cell cycle of astrocyte in vitro.Methods:We measured the cell cycles and the proliferation of astrocyte in different time after ischemia and anoxia by flow cytometry and Brdu pulse labeling; and detected the expression of GFAP,PCNA and cyclinD1 with the fluorescence immunocytochemistry.Result:After ischemia and anoxia in vitro,S phase of astrocyte significantly increased compared with the normal,and the peak appeared at 6h;and the proliferated ability of astrocyte is highest at 6h by Brdu pulse labeling test,but the S phase and proliferated ability is decreased after 6h.At the early of ischemia and anoxia,the positive staining of GFAP increased;but after ischemia and anoxia 12 hours,the positive staining of GFAP is weak,The levels of PCNA increased after the damage,and reached the peak at 6 h.The expression of cyclinD1 is gradually increased after damaged,the peak is appeared at 24h.Conclusion:Astrocyte cells are activated to proliferate and enter into new cell cycle events by ischemia and anoxia;PCNA and cyclinD1 counsel the adjustment of astrocyte’s proliferation and repairing;The cell cycle events are related with proliferation and activation of astrocyte closely.更多还原