【作者】 冷洁；

【导师】 蒋莉；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 博士

【摘要】 第一部分持续惊厥后幼年鼠脑内神经发生的动态变化和迁移趋势目的:探讨惊厥持续状态（status convulsion, SC）后未成熟大鼠脑内神经发生的动态变化。方法:建立幼年Wistar鼠3h SC模型,在SC后0天至40天的7个时间点上处死动物,处死前1天腹腔注射5-溴2-脱氧尿嘧啶核苷（5-bromo 2-deoxyuridine, BrdU）;采用免疫组化方法动态检测海马、胼胝体下区（subcallosal zone, SCZ）和皮层在各时间点BrdU的表达,确定SC后各时间点上脑内不同区域神经干细胞（neural stem cells, NSCs）的增殖水平和迁移趋势。结果:⑴正常未成熟脑海马齿状回（dentate gyrus,DG）的颗粒细胞下层（subgranular zone,SGZ）和胼胝体下区（subcallosal zone,SCZ）以及皮层都存在少量BrdU阳性细胞;与对照组相比,SC后0天海马DG的SGZ,海马CA1、CA3区,SCZ以及皮层BrdU阳性细胞即开始显著升高,海马的SGZ、CA1、CA3以及皮层于SC后2天出现增殖高峰,SCZ于SC后7天出现增殖高峰。⑵SC后2天DG分子层出现BrdU阳性细胞,SC后7天DG门区出现BrdU阳性细胞。SGZ新生细胞有向DG分子层迁移趋势,SCZ细胞有向CA1、CA3和皮层迁移趋势。结论:SC刺激未成熟脑内神经发生,部分新生细胞发生异位迁移,部分细胞可能向海马神经细胞损伤区迁移。第二部分体外培养大鼠海马神经干细胞分化前与分化后电压门控钠、钾通道的表达目的:观察体外分离培养大鼠海马神经干细胞（neural stem cells, NSCs）分化前后钠、钾离子通道的表达。方法:无血清培养体外分离、纯化孕15-16天Wistar胎鼠的海马NSCs,nestin免疫荧光染色鉴定培养细胞是否为干细胞,无血清促分化培养基促分化,β-III-tubulin、GFAP荧光鉴定分化细胞类型。显微注射荧光黄标记记录细胞,采用β-III-tubulin免疫荧光染色检测所记录细胞是否具有神经元表型。膜片钳记录NSCs分化前和分化后具有神经元样细胞的电压门控钠、钾离子通道的表达,结果:⑴体外培养的海马NSCs nestin免疫染色阳性,具有自我增殖能力能在体外诱导分化为神经元或胶质细胞;⑵膜片钳记录的细胞均具有神经元表型;⑶未分化NSCs无内向性钠电流,分化后1天即可检测到内向性钠电流;未分化和分化的NSCs均表达瞬时外向钾电流和外向延迟整流钾电流。结论:⑴采用无血清培养方法,在体外成功分离海马NSCs;⑵神经发生过程中钠通道的功能性表达可能是NSCs退出细胞周期开始分化的标志。未分化和分化的NSCs表达两种类型的外向性钾离子电流。第三部分脑源性神经营养因子对体外培养大鼠海马神经干细胞分化过程中被动膜特性和电压门控钠、钾通道电生理特征的影响目的:探讨脑源性神经营养因子（brain derived neurotrophic factor, BDNF）对海马神经干细胞（neural stem cells, NSCs）被动膜特性和电压门控钠、钾通道电生理特征的影响方法:采用无血清促分化培养基促NSCs体外分化,选择体外分化前期（1-4天）和体外分化后期（8-15天）具有神经元形态细胞进行膜片钳记录;记录细胞被动膜特性、钠电流,分析钠电流密度,拟合钠通道激活曲线,计算半数激活电压和斜率因子。记录总钾电流,分离瞬时外向钾电流和外向延迟整流钾电流;分析两种电流在分化前期和后期的表达情况;分析电流密度,拟合激活曲线,计算半数激活电压和斜率因子。结果:⑴NSCs分化早期,BDNF促进静息电位左移,膜电容增加,输入阻抗降低,时间常数延长。⑵无BDNF干预,对照组钠电流密度随分化逐渐增加;BDNF干预后,分化前期,BDNF作用组钠电流密度明显高于对照组;而分化后期BDNF作用组钠电流密度明显低于对照组;BDNF长期作用使钠电流激活曲线右移。⑶所有记录的细胞都表达I_K（DR） ;无BDNF干预,对照组I_K（A）的表达随着分化逐渐增加;BDNF干预后,I_K（A）的表达随着分化逐渐降低;分化前期,BDNF作用组总钾电流密度较对照组明显增加;分化后期,两个实验组的总钾电流密度没有差异;无BDNF干预,随着分化对照组I_K（A）和I_K（DR）半数激活电压都发生左移;分化前期,与对照组相比I_K（A）和I_K（DR）半数激活电压也都发生左移,分化后期,BDNF作用组与对照组之间没有差异。结论:⑴分化前期为细胞被动膜特性发育的关键时期,BDNF在NSCs细胞形态改变的同时促进NSCs来源的神经元样细胞被动膜特性发育,以促进NSCs被动膜特性趋向成熟化。⑵体外培养NSCs分化前期（1-4天）也是细胞钠、钾通道发育的关键时期;⑶BDNF促进分化早期钠通道的表达和/或开放,而长期作用则抑制钠通道的表达和/或开放;使钠通道激活曲线右移,具有抑制新生神经细胞兴奋性作用。⑷BDNF与I_K（A）的表达密切相关,BDNF可能抑制I_K（A）的表达,I_K（A）在钾通道的发育过程中具有一定作用;BDNF促进分化早期钾通道激活曲线左移,钾电流密度增加,促进钾通道的表达和/或开放。⑸BDNF参与调节NSCs钠、钾离子通道发育关键时期,降低神经元样细胞兴奋性,对惊厥后的产生的神经细胞可能起保护作用。更多还原

【Abstract】 PART I PROLONGED SEIZURES INCREASE PROLIFERATING NEUROBLASTS IN THE IMATURE RAT HIPPOCAMPUS, SUBCALLOSAL ZONE, CORTEX AND THE TRENDS OF MIGRATIONObjective: To explore the dynamic changes of neurogenesis after status convulsion in the immature rat brain.Method: Rats were induced by chemoconvulsants lithium- pilocarpine and were killed 4h, 2 ,7,13,20,27,40d afer. All rats received four injections of bromodeoxyuridine （BrdU） with 2-h intervals 1 day before killed. Animals were perfused and brains were processed for immunocytochemistry antibodies against BrdU. Comparisons of the number of nuclei positive for BrdU which is the marker of proliferating cells at the 7 time points of hippocampus, subcallosal zone and cortex were performed to explore the proliferation and the trends of migration.Results: Our experiments with the lithium-pilocarpine models suggested that acute seizures considerably increase neurogenesis of subgranular zone （SGZ）, subcallosal zone （SCZ） and cortex immediately when compared with the age-matched-control groups. Only a few of cells labeled by BrdU were found in SGZ, SCZ and cortex in the age-matched-control groups. SC induced a transitory proliferative surge in the SGZ、CA1、CA3 and cortex with the number of new neurons increasing several folds 2 days after SC which had the trend to migrate to the molecular layer and hilar of the dentate gyrus （DG）, but 7 days after in the SCZ with the tendency of migrating toward CA1、CA3 and cortex.Conclusion: Prolonged seizure activity markedly increases neurogenesis of immature brain. Seizure recruits new born cells into abnormal locations and injured hippocampus.PART II THE EXPRESSION CHARACTERISTICS OF VOLTAGE-GATED SODIUM AND POTASSIUM CHANNELS VIA CULTURED NEURAL STEM CELLS DIFFERENTIATION.Objective: To exam the expression of voltage-gated sodium and potassium channels of neural stem cells cultured in vitro before and after differentiation.Method: Embryonic rat hippocampal neural stem cells（NSCs） were isolated and cultured in serum-free medium. Use nestin immunofluorescence labeling to identify if the cultured cells were neural stem cells. Passaged cells were plated on poly-llysine hydrochloride glass coverslips for 1 hour or differentiated for 1 to 3 days with differentiation serum-free medium. Useβ-III-tubulin and GFAP immunofluorescence labeling to identify the fate of these cells. Whole cell patch-clamp technique was used to record the voltage-gated ion channel currents in the neural like cells. To permit further visualization of the cell morphology, cells were routinely filled with Lucifer yellow （LY） during the recording, then colocalized withβ-III-tubulin to identify if the test cells were neuronal progenitors or neurons.Results: Cells aggregated could proliferate and form neurospheres which nestin immunoreactivity was within 3-5 days after primary culture. NSCs have been shown to differentiate into neurons, astrocytes in vitro. No inward Na+ current was detected in any of the undifferentiated NSCs.1 d after the induction of differentiating, cells exhibited voltage-gated sodium currents. The presence of two types of outward potassium currents, delayed recitifier potassium currents and transient potassium currents were also recorded before or after differentiation. Conclusion: NSCs from the embryonic rat brain could be isolated, cultured and differentiated. sodium current expression is a very early event following cell cycle exit in neurogenesis. The two types of outward potassium currents expressed either before or after differentztion.PART III MODULATION OF PASSIVE MEMBRANE PROPERTIES, VOLTAGE-GATED SODIUM CHANNELS AND VOLTAGE-GATED POTASSIUM CHANNELS BY BRAIN-DERIVED NEUROTROPHIC FACTOR DURING DIFFERENTIATIONObjective: To explore the brain derived neurotrophic factor （BDNF） modulation of passive membrane properties and the electrophysiological properties of voltage-gated sodium and potassium channels during differentiation.Method: Passaged cells were plated on poly-llysine hydrochloride glass coverslips with differentiation serum-free medium. Whole cell patch clamp recordings were used to monitor the passive membrane properties,sodium and potassium currents. Analyze the passive membrane properties、sodium current density, generated the activation curves, compared the mean half activation voltage （V1/2） and mean slope k at the early development stage （DIV 1-4） and the late development stage （DIV 8-14）.Analyze the potassium current density. Different holding membrane potentials were used to identify the two current types: delayed recitifier potassium currents and transient potassium currents.generated the activation curves, compared the mean half activation voltage (V_1/2) and mean slope k at the early development stage （DIV 1-4） and the late development stage （DIV 8-14）.Results: Compared with the time-match controls, Cells treated with BDNF were accompanied by a gradual increase in membrane capacitance, time constant and decrease in input resistance during the progress of development. Chronic stimulation of neuron-like cell cultures with BDNF evoked biphasic changes in sodium currents (I_dNa). During the early stage of differentiation chronic treatment with BDNF induced a significantly increase of sodium current density, but evoked opposite changes at the later stage, compared with the time-match control group.Chronic BDNF stimulation revealed an increase in total whole-cell potassium current density in BDNF-treated neurons at the early development stage and no significant difference at the later development stage compared with time-match controls. The delayed rectifier outward currents are observed in all neuron-like cells. The expression of I_K（A） increased significantly following induction of neural differentiation treated without BDNF, but the expression of I_K（A） decreased after the supplementation of BDNF. Current–conductance relationships indicated that there was negative shift in the V_1/2 of I_K（A） and I_K（DR） following chronic exposure of neuron-like cells to BDNF during the early stage of differentiation but no significant change of the mean slope during the two differentiate stages compared with time-match controls.Conclusion: significant changes in passive membrane properties accompany the dramatic morphological transition observed during the different differentiated stages of cells treated with BDNF. The early stage of differentiation might be a critical period, BDNF was involved in the regulation of the critical period by promoting the functional development of passive membrane. The difference of I_dNa between the early stage of differentiation and the late stage of differentiation stimulated by BDNF indicated that BDNF might play an important role by regulating the formation and/or activation of sodium channels. Our results strongly indicated that the early development stage （1-4days） was a critical period for the development of sodium channels and BDNF could play a protective role in the epileptic brain which reduced excitatory of the new born cells. BDNF might play an important role by regulating the formation and/or activation of potassium channels at the early development stage. There was a significant correlation between the supplementation of BDNF and the expression of I_K（A）. I_K（A） might be suppressed by BDNF and might serve a developmental function during the period when it is present.The early development stage （DIV 1-4） might be a critical period of potassium channels during which BDNF might be involved in regulating potassium channels and served to limit excitability in neural cells.更多还原