重组腺相关病毒神经肽Y基因转染对海人酸致痫大鼠海马突触重建的影响及其机制研究
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摘要
癫痫是由各种原因导致的脑细胞群异常放电所致的突然性、反复性和短暂性的神经功能失常为特征的综合征。中国目前至少有700多万癫痫患者，在美国也大约有80多万人在忍受药物难治性癫痫的疾病折磨。在过去的30年里，全世界有近30000中药物被用来进行癫痫治疗的动物实验，以此来提高人类临床治疗癫痫的发展，多年的研究已经取得了良好的效果，使一部分癫痫患者通过药物治疗能够得到缓解，但仍有部分癫痫患者对药物治疗无效，或者不能阻止这些药物难治性癫痫的发作和进展。目前药物难治性癫痫的外科手术治疗，也不能完全改变脑内神经细胞的高兴奋状态和相关离子通道的异常，同时，外科手术的局限性仍然只能使少部分癫痫患者从中受益，大部分患者仍然依赖长期的规范的药物治疗。随着对该癫痫发病机制研究的进一步深入，以及分子生物学技术的发展，基因治疗作为一种新的治疗手段为有效控制和彻底治愈癫痫带来了新的希望。
在对癫痫发病机制的研究中，海马突触重建被认为与颞叶癫痫发作关系密切，而在海马异位突触的重建过程中，苔藓纤维出芽（mossy fibersprouting, MFS）是突触重建的重要标志，同时伴随突触素（synaptophysinP38）以及突触生长相关蛋白的异常表达，而这种异位突触的形成又反过来加重了癫痫的发作。
在对突出重建后新生突触的超微结构研究中发现，新生突触在癫痫持续状态下，新生突触的形态、数量、结构、以及功能均区别于正常脑组织的神经突触，这些新生的突触形成的是兴奋性神经环路还是抑制性环路，也受到了广泛关注。显微结构显示这些新生的异位突触大部分是非对称性突触，而这是典型的兴奋性突触的特征。因此苔藓纤维出芽与颗粒细胞形成的异位突触被认为是诱发癫痫发作的重要结构基础。
在对癫痫药物治疗多年的研究中发现，神经肽Y(neuropeptide Y, NPY)作为神经递质和神经调质广泛存在于中枢神经系统和周围神经系统，中枢神经系统中海马内浓度最高。NPY及其受体与癫痫的发生以及苔藓纤维出芽关系密切，且外源性NPY具有抗癫痫作用。但NPY在癫痫模型中对神经功能的作用非常复杂，常因动物模型不同或给药途径的不同出现不一致甚至相互矛盾的实验结果。
本研究以重组腺相关病毒作为载体，将NPY基因转染到脑组织特定靶区，观察其对海人酸（Kainic acid, KA）致痫大鼠癫痫发作的影响，并进一步运用Timm染色、PCR、免疫印迹等方法，观察分析重组腺相关病毒基因转染的NPY（rAAV2/1-NPY-EGFP）对海马CA3区苔藓纤维出芽、突触素表达、突触生长相关蛋白（GAP-43）的影响，以及利用透射电镜观察rAAV2/1-NPY-EGFP治疗对致痫大鼠海马突触超微结构的影响。
第一部分重组腺相关病毒神经肽Y基因转染对海人酸致痫大鼠癫痫发作及海马苔藓纤维出芽的影响
目的：探讨重组腺相关病毒神经肽Y（rAAV2/1-NPY-EGFP）基因转染对癫痫发作程度和海马苔藓纤维出芽的影响。
方法：清洁级健康Wistar大鼠30只，体重250～270g，随机分成NPY实验组（n=12），海人酸致痫组（KA）（n=12）和对照组（n=6）。NPY实验组：脑室注射10μl的rAAV2/1-NPY-EGFP，滴度为5×1011μg/ml；KA组在脑室内注射等量的生理盐水。以上两组10min后分别在海马CA3区注射海人酸（KA）；空白对照组依次分别在脑室和CA3区注射等量的生理盐水。并分别于注射后2、4周观察大鼠的癫痫发作情况，视频脑电（EEG）癫痫波的频率和波幅，Timm染色观察海马CA3区苔藓纤维出芽。
结果：
1NPY实验组大鼠随观察时间的延长，发作程度逐渐减轻，与KA组大鼠相比，于4W时，脑电图癫痫波放电频率减少(P <0.05)，波幅降低(P<0.05)。
2NPY实验组苔藓纤维出芽分级明显低于对照组。3.空白对照组没有癫痫发作，CA3区苔藓纤维出芽没有明显变化。
结论：rAAV2/1-NPY–EGFP基因转染可以降低海马CA3区苔藓纤维出芽程度，有效抑制癫痫发作，为NPY基因治疗临床难治性癫痫提供了有力试验支持。
第二部分重组腺相关病毒神经Y基因转染对海人酸致痫大鼠海马突触素表达的影响
目的：观察rAAV2/1-NPY-EGFP基因转染对突触素表达的影响，探讨癫痫治疗的新方法
方法：清洁级健康Wistar大鼠，体重250～270g，随机分成NPY实验组（n=24），海人酸致痫组（KA组）（n=24）和对照组（n=24）。NPY实验组：脑室注射10μl的rAAV2/1-NPY-EGFP，滴度为5×1011μg/ml；海人酸致痫组和对照组分别在脑室内注射等量的生理盐水。10min后分别在NPY试验组及海人酸致痫组大鼠的海马CA3区注射海人酸（Kainic acid,KA）；对照组在CA3区注射等量的生理盐水。注射后2、4周三组大鼠分别取12只，断头取海马组织，分别应用荧光定量PCR和western-blot方法观察突触素（P38）表达的变化。
结果：
1NPY实验组大鼠与海人酸致痫组（KA组）大鼠相比，4W后前者发作程度明显减轻，差异有显著性，对照组无癫痫发作。
24W后突触素（P38）表达的PCR结果和Western-blot结果显示，在NPY实验组和海人酸致痫组大鼠之间存在明显差异(P <0.05)，而2W时差异性不明显。
结论：rAAV2/1-NPY–EGFP基因转染可以降低海马突触素（P38）的表达，从而抑制突触重建的发生，有效抑制癫痫的发作。第三部分重组腺相关病毒神经Y基因转染对海人酸致痫大鼠海马突触生长相关蛋白GAP-43表达的影响
目的：观察rAAV2/1-NPY-EGFP基因转染对突触生长相关蛋白GAP-43表达的影响，探讨癫痫治疗的新方法
方法：清洁级健康Wistar大鼠，体重250～270g，随机分成NPY实验组（n=48），海人酸致痫组（KA组）（n=48）和对照组（n=48）。NPY实验组：脑室注射10μl的rAAV2/1-NPY-EGFP，滴度为5×1011μg/ml；海人酸致痫组和对照组分别在脑室内注射等量的生理盐水。10min后分别在NPY试验组及海人酸致痫组大鼠的海马CA3区注射海人酸（Kainic acid, KA）；对照组在CA3区注射等量的生理盐水。注射后2、4周三组大鼠分别取12只，断头取海马组织，分别应用免疫组化学方法和westen-blot方法观察GAP-43表达的变化。
结果：NPY实验组大鼠与海人酸致痫组（KA组）大鼠相比，4W后突触生长相关蛋白GAP-43表达的PCR结果和Western-blot结果显示，在NPY实验组和海人酸致痫组大鼠之间存在明显差异(P <0.05)，而2W时差异性不明显。
结论：rAAV2/1-NPY–EGFP基因转染4W后降低了海马突触生长相关蛋白GAP-43的表达，从而抑制了突触重建的发生。
第四部分重组腺相关病毒神经Y基因转染对海人酸致痫大鼠海马苔藓纤维出芽突触超微结构的影响
目的：探讨重组腺相关病毒基因转染NPY对苔藓纤维出芽形成的突触超微结构的影响。
方法：清洁级健康Wistar大鼠，体重250～270g，随机分成NPY实验组（n=24），海人酸致痫组（KA组）（n=24）和对照组（n=24）。NPY实验组：脑室注射10μl的rAAV2/1-NPY-EGFP，滴度为5×1011μg/ml；海人酸致痫组和对照组分别在脑室内注射等量的生理盐水。10min后分别在NPY试验组及海人酸致痫组大鼠的海马CA3区注射海人酸（Kainic acid,KA）；对照组在CA3区注射等量的生理盐水。分别于注射后2、4周取海马组织；采用Timm染色标记出芽苔藓纤维末端，在电镜下观察新生的突触数量、突触囊泡密度、以及突触间隙变化。
结果：
1海人酸致痫后苔藓纤维出芽形成的新生突触主要以非对称性突触为主，非对称性突触数量增加，重组腺相关病毒NPY基因转染后4W，非对称性突触数量减少，与致痫组对比，差异有显著性；
2海人酸致痫组突触间隙模糊增宽，重组腺相关病毒NPY基因转染后4W，突触间隙接近正常，两组相对比差异有显著性；
3海人酸致痫组突触囊泡突触囊泡减少，形态不规则，密度不均匀，重组腺相关病毒NPY基因转染后4W，突触囊泡数密度（synaptic vesiclesdensity, SVD）接近正常，与致痫组对比差异有显著性；
4海人酸致痫组以及NPY基因转染后2W神经元在电镜下显示为：细胞核肿胀、核仁消失；线粒体空泡变性、线粒体嵴和膜溶解、分界不清；粗面内质网脱颗粒现象，以上数据和对照组以及NPY基因转染4W组相比，差异有显著性（P＜0.05），海人酸致痫组和NPY基因转染组2W之间对比，无显著性差异。
结论：非对称性突触是海人酸致痫大鼠海马苔藓纤维出芽新生突触的主要类型，这种新生的突触类型兴奋性增加，NPY能够抑制新生的非对称突触的形成，从而抑制癫痫的发生。KA致痫大鼠海马神经元细胞凋亡明显增加，基因转染NPY对神经细胞的凋亡有抑制作用。
Epilepsy is a brain dysfunctional syndrome which characteristics issudden, recurrent and transient neurological disorder caused by abnormaldischarge of neurons with various reasons. There are about700Chinese withepilepsy currently, and this condition afflicts at least800000Americans also.Over the past30years, there are about30000drugs were used to carry outepilepsy treatment in animal experiments all over the world, it has achievedgood effect, many epilepsy patients can get relief through the drug therapy,unfortunation, pharmacoyherapy usually fails to achieve long-term remission,it can not inhibit the seizure or recurrent. Currently, the surgery is mainlytaken to treat the refractory epilepsy to resect the epileptic zone, it can notchange the neuronal hyperexcitability and abnormal function of ion channelcompletely, most patients still rely on long-term standard pharmacoyherapy.with the pathogenesis and molecular biology technology developing, the genetherapy as a new method to control and treat epilepsy effectively will bringinnovative and promising alternative.
In the pathogenesis research of epilepsy, it is considered that thehippocampal synaptic reorganization associated with temporal lobe epilepsyclosely, and in hippocampal synaptic reorganization process, moss fibersprouting (MFS) is an important symbol of synaptic reconstruction, and at thesame time with synaptophysin (P38) and synaptic growth-associated protein43(GAP-43) abnormal expressing, this kind of synapse formation reverse toaggratate epilepsy seizures.
The synaptic ultrastructural study found that the morphology, quantity,structure, and function of new synapses in status epilepticus, are different from normal brain neural, these new synapse formation is excitatory circuitry orinexcitatory circuitry, also have aroused wide public attention. The study ofsynaptic ultrastructural shows that most of these recurrent synaptic isasymmetry synapses, and this is a typical characteristics of excitabilitysynaptic. So moss fiber sprouting and granular cell formation of ectopicsynapse is considered to be the important basic structure induced to seizures.
The study of pharmacoyherapy in epilepsy over many years,neuropeptide Y (NPY) as a neurotransmitter and neuromodulator wide spreadover the central nervous system and peripheral nervous system, and it has thehighest concentration in the hippocampus of central nervous system. NPY andits receptors have the relationship with the epileptogenesis and moss fibersprouting, and exogenous NPY has antiepileptic effect. But the neural functionof NPY is very complex in epilepsy model, it appear inconsistent and evenconflicting experimental results with different animal model or differentmethod of administration.
In this study, NPY gene carried by the recombinant adeno-associatedvirus as a vector, is transfered to specific target area of brain, the influence ofNPY gene to epileptic seizure rats which induced by Kainic acid (KA) wasobserved about the MFS, synaptic element expression, synaptic growth-associated protein (GAP-43) in hippocampal CA3using Timm dyeing, PCR,western blot method, and the influence of rAAV2/1-NPY-EGFP onhippocampal synaptic ultrastructural using transmission electron microscope.
Part1The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on seizure and mossy fiber sprouting inhippocampus in rat model induced by kainic acid
Objective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on seizure and mossy fibersprouting in hippocampus in rat.
Methods:30clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=12), KA group (n=12) and controlgroup(n=6). NPY group in which10μl of rAAV2/1-NPY-EGFP (titer5× 1011v.g./ml) were injected to ventricle, KA group, in which amount saline wasinjected to ventricle. Ten minutes later, KA were injected respectively tohippocampal CA3area of two groups, The control group respectively wereinjected in ventricle and CA3area of same amount of saline for two times.Theseizure situation, EEG wave frequency and amplitude were observed afterinjection of2and4week. Mossy fiber sprouting(MFS) in the hippocampuswere detected by Timm’s staining after injection of4w.
Result:
1The seizure degree in rats of NPY group gradually reduced withobservation time. At4W post-injection, in rats of NPY group, the onset ofsymptoms and EEG epileptic discharge frequency and amplitude decreased(P<0.05), compared to rats of positive control group.
2The scores of mossy fiber sprouting in the hippocampal CA3area werehigher in KA group compareed to NPY group.3The control group had noseizure and no significant difference of mossy fiber sprouting.
Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the hippocampal MFS, which provide strong support to the NPYgene therapy for clinical refractory epilepsy.
Part2The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on expression of synaptophysin inhippocampus in rat model induced by kainic acid
Objective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on expression of synaptophysin inhippocampus in rat.
Methods: Clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=24), KA group (n=24) and controlgroup(n=24). NPY group in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle, KA group, in which amountsaline was injected to ventricle. Ten minutes later, KA were injectedrespectively to hippocampal CA3area of two groups, The control grouprespectively were injected in ventricle and CA3area of same amount of saline for two times. Three groups were taken the hippocampal tissue, to observedthe changes of expression of synaptophysin after injection of2and4weekwhich experimentation was performed by fluorescent quantitative PCR andwesten-blot.
Result:
1The seizure degree in rats of NPY groups decreased significantly after4W injection compared with KA groups (P<0.05).
2The results of PCR and Westen-blot on synaptophysin (P38)expression showed that there are obvious difference between NPY groups andKA groups after4W(P <0.05), and there are no difference after2W.
Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the synaptophysin (P38) expression, thus to inhibit epilepsy.
Part3The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on expression of synaptic growth-associatedprotein43(GAP-43) in hippocampus in rat model induced by kainic acid
Objective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on expression of synapticgrowth-associated protein43(GAP-43) in hippocampus in rat.
Methods: Clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=48), KA group (n=48) and controlgroup(n=48). NPY group in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle, KA group, in which amountsaline was injected to ventricle. Ten minutes later, KA were injectedrespectively to hippocampal CA3area of two groups, The control grouprespectively were injected in ventricle and CA3area of same amount of salinefor two times. Three groups were taken the hippocampal tissue, to observedthe changes of expression of GAP-43after injection of2and4week whichexperimentation was performed by immunohistochemical and fluorescentquantitative PCR.
Result: The results of immunohistochemical and fluorescent quantitativePCR on GAP-43expression showed that there are obvious difference between NPY groups and KA groups after4W(P<0.05), and there are no differenceafter2W.
Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the GAP-43expression, thus to inhibit the synaptic reorganization.
Part4The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on ultrastructure features of MFS inhippocampus in rat model induced by kainic acid
Objective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on ultrastructure features of MFSin hippocampus in rat.
Methods: Clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=24), KA group (n=24) and controlgroup(n=24). NPY group in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle, KA group, in which amountsaline was injected to ventricle. Ten minutes later, KA were injectedrespectively to hippocampal CA3area of two groups, The control grouprespectively were injected in ventricle and CA3area of same amount of salinefor two times. Three groups were taken the hippocampal tissue, mossy fibersprouting(MFS) in the hippocampus were detected by Timmhistochemistry,to observed the changes of the synaptic quantity, synapticvesicles density, and the synaptic cleft under the electron microscope.
Result:
1Most of the mossy fiber sprouting synaptic terminals were asym-metric synapses, the quantity of asymmetric synapses increased in KA groupand NPY group after2W, the quantity of asymmetric synapses decreased inNPY group after4W, there are obvious difference between NPY groups andKA groups after4W(P <0.05).
2The synaptic cleft increased in KA group and NPY group after2W, anddecreased in NPY group after4W, there are obvious difference between twogroups after4W(P <0.05).
3It is showed that the quantity of synaptic vesicles reducing, shape of synaptic vesicles being irregular, and the density of synaptic vesicles beingreorganization in KA group and NPY group after2W. The synaptic vesiclesdensity(SVD) is normal after4W, there are obvious difference between NPYgroups and KA groups after4W(P <0.05).
4It is showed that the nucleus swelling, entoblast disappearing, mitoch-ondrial vacuolar degeneration, mitochondrial ridge and membrane dissolved,and the rough endoplasmic reticulum degranulation under the electronmicroscope in KA group and NPY group after2W, comparing to NPY groupafter4W, there are significant difference(P <0.05).
Conclusion: Most of the mossy fiber sprouting synaptics terminals areasymmetric synapses, this kind of new synaptic is irritability, and can beinhibited by NPY. The apoptosis of neuron in hippocampal increased in rat ofepilepsy, and this apoptosis of neuron can be inhibited by NPY.
在对癫痫发病机制的研究中，海马突触重建被认为与颞叶癫痫发作关系密切，而在海马异位突触的重建过程中，苔藓纤维出芽（mossy fibersprouting, MFS）是突触重建的重要标志，同时伴随突触素（synaptophysinP38）以及突触生长相关蛋白的异常表达，而这种异位突触的形成又反过来加重了癫痫的发作。
在对突出重建后新生突触的超微结构研究中发现，新生突触在癫痫持续状态下，新生突触的形态、数量、结构、以及功能均区别于正常脑组织的神经突触，这些新生的突触形成的是兴奋性神经环路还是抑制性环路，也受到了广泛关注。显微结构显示这些新生的异位突触大部分是非对称性突触，而这是典型的兴奋性突触的特征。因此苔藓纤维出芽与颗粒细胞形成的异位突触被认为是诱发癫痫发作的重要结构基础。
在对癫痫药物治疗多年的研究中发现，神经肽Y(neuropeptide Y, NPY)作为神经递质和神经调质广泛存在于中枢神经系统和周围神经系统，中枢神经系统中海马内浓度最高。NPY及其受体与癫痫的发生以及苔藓纤维出芽关系密切，且外源性NPY具有抗癫痫作用。但NPY在癫痫模型中对神经功能的作用非常复杂，常因动物模型不同或给药途径的不同出现不一致甚至相互矛盾的实验结果。
本研究以重组腺相关病毒作为载体，将NPY基因转染到脑组织特定靶区，观察其对海人酸（Kainic acid, KA）致痫大鼠癫痫发作的影响，并进一步运用Timm染色、PCR、免疫印迹等方法，观察分析重组腺相关病毒基因转染的NPY（rAAV2/1-NPY-EGFP）对海马CA3区苔藓纤维出芽、突触素表达、突触生长相关蛋白（GAP-43）的影响，以及利用透射电镜观察rAAV2/1-NPY-EGFP治疗对致痫大鼠海马突触超微结构的影响。
第一部分重组腺相关病毒神经肽Y基因转染对海人酸致痫大鼠癫痫发作及海马苔藓纤维出芽的影响
目的：探讨重组腺相关病毒神经肽Y（rAAV2/1-NPY-EGFP）基因转染对癫痫发作程度和海马苔藓纤维出芽的影响。
方法：清洁级健康Wistar大鼠30只，体重250～270g，随机分成NPY实验组（n=12），海人酸致痫组（KA）（n=12）和对照组（n=6）。NPY实验组：脑室注射10μl的rAAV2/1-NPY-EGFP，滴度为5×1011μg/ml；KA组在脑室内注射等量的生理盐水。以上两组10min后分别在海马CA3区注射海人酸（KA）；空白对照组依次分别在脑室和CA3区注射等量的生理盐水。并分别于注射后2、4周观察大鼠的癫痫发作情况，视频脑电（EEG）癫痫波的频率和波幅，Timm染色观察海马CA3区苔藓纤维出芽。
结果：
1NPY实验组大鼠随观察时间的延长，发作程度逐渐减轻，与KA组大鼠相比，于4W时，脑电图癫痫波放电频率减少(P <0.05)，波幅降低(P<0.05)。
2NPY实验组苔藓纤维出芽分级明显低于对照组。3.空白对照组没有癫痫发作，CA3区苔藓纤维出芽没有明显变化。
结论：rAAV2/1-NPY–EGFP基因转染可以降低海马CA3区苔藓纤维出芽程度，有效抑制癫痫发作，为NPY基因治疗临床难治性癫痫提供了有力试验支持。
第二部分重组腺相关病毒神经Y基因转染对海人酸致痫大鼠海马突触素表达的影响
目的：观察rAAV2/1-NPY-EGFP基因转染对突触素表达的影响，探讨癫痫治疗的新方法
方法：清洁级健康Wistar大鼠，体重250～270g，随机分成NPY实验组（n=24），海人酸致痫组（KA组）（n=24）和对照组（n=24）。NPY实验组：脑室注射10μl的rAAV2/1-NPY-EGFP，滴度为5×1011μg/ml；海人酸致痫组和对照组分别在脑室内注射等量的生理盐水。10min后分别在NPY试验组及海人酸致痫组大鼠的海马CA3区注射海人酸（Kainic acid,KA）；对照组在CA3区注射等量的生理盐水。注射后2、4周三组大鼠分别取12只，断头取海马组织，分别应用荧光定量PCR和western-blot方法观察突触素（P38）表达的变化。
结果：
1NPY实验组大鼠与海人酸致痫组（KA组）大鼠相比，4W后前者发作程度明显减轻，差异有显著性，对照组无癫痫发作。
24W后突触素（P38）表达的PCR结果和Western-blot结果显示，在NPY实验组和海人酸致痫组大鼠之间存在明显差异(P <0.05)，而2W时差异性不明显。
结论：rAAV2/1-NPY–EGFP基因转染可以降低海马突触素（P38）的表达，从而抑制突触重建的发生，有效抑制癫痫的发作。第三部分重组腺相关病毒神经Y基因转染对海人酸致痫大鼠海马突触生长相关蛋白GAP-43表达的影响
目的：观察rAAV2/1-NPY-EGFP基因转染对突触生长相关蛋白GAP-43表达的影响，探讨癫痫治疗的新方法
方法：清洁级健康Wistar大鼠，体重250～270g，随机分成NPY实验组（n=48），海人酸致痫组（KA组）（n=48）和对照组（n=48）。NPY实验组：脑室注射10μl的rAAV2/1-NPY-EGFP，滴度为5×1011μg/ml；海人酸致痫组和对照组分别在脑室内注射等量的生理盐水。10min后分别在NPY试验组及海人酸致痫组大鼠的海马CA3区注射海人酸（Kainic acid, KA）；对照组在CA3区注射等量的生理盐水。注射后2、4周三组大鼠分别取12只，断头取海马组织，分别应用免疫组化学方法和westen-blot方法观察GAP-43表达的变化。
结果：NPY实验组大鼠与海人酸致痫组（KA组）大鼠相比，4W后突触生长相关蛋白GAP-43表达的PCR结果和Western-blot结果显示，在NPY实验组和海人酸致痫组大鼠之间存在明显差异(P <0.05)，而2W时差异性不明显。
结论：rAAV2/1-NPY–EGFP基因转染4W后降低了海马突触生长相关蛋白GAP-43的表达，从而抑制了突触重建的发生。
第四部分重组腺相关病毒神经Y基因转染对海人酸致痫大鼠海马苔藓纤维出芽突触超微结构的影响
目的：探讨重组腺相关病毒基因转染NPY对苔藓纤维出芽形成的突触超微结构的影响。
方法：清洁级健康Wistar大鼠，体重250～270g，随机分成NPY实验组（n=24），海人酸致痫组（KA组）（n=24）和对照组（n=24）。NPY实验组：脑室注射10μl的rAAV2/1-NPY-EGFP，滴度为5×1011μg/ml；海人酸致痫组和对照组分别在脑室内注射等量的生理盐水。10min后分别在NPY试验组及海人酸致痫组大鼠的海马CA3区注射海人酸（Kainic acid,KA）；对照组在CA3区注射等量的生理盐水。分别于注射后2、4周取海马组织；采用Timm染色标记出芽苔藓纤维末端，在电镜下观察新生的突触数量、突触囊泡密度、以及突触间隙变化。
结果：
1海人酸致痫后苔藓纤维出芽形成的新生突触主要以非对称性突触为主，非对称性突触数量增加，重组腺相关病毒NPY基因转染后4W，非对称性突触数量减少，与致痫组对比，差异有显著性；
2海人酸致痫组突触间隙模糊增宽，重组腺相关病毒NPY基因转染后4W，突触间隙接近正常，两组相对比差异有显著性；
3海人酸致痫组突触囊泡突触囊泡减少，形态不规则，密度不均匀，重组腺相关病毒NPY基因转染后4W，突触囊泡数密度（synaptic vesiclesdensity, SVD）接近正常，与致痫组对比差异有显著性；
4海人酸致痫组以及NPY基因转染后2W神经元在电镜下显示为：细胞核肿胀、核仁消失；线粒体空泡变性、线粒体嵴和膜溶解、分界不清；粗面内质网脱颗粒现象，以上数据和对照组以及NPY基因转染4W组相比，差异有显著性（P＜0.05），海人酸致痫组和NPY基因转染组2W之间对比，无显著性差异。
结论：非对称性突触是海人酸致痫大鼠海马苔藓纤维出芽新生突触的主要类型，这种新生的突触类型兴奋性增加，NPY能够抑制新生的非对称突触的形成，从而抑制癫痫的发生。KA致痫大鼠海马神经元细胞凋亡明显增加，基因转染NPY对神经细胞的凋亡有抑制作用。
Epilepsy is a brain dysfunctional syndrome which characteristics issudden, recurrent and transient neurological disorder caused by abnormaldischarge of neurons with various reasons. There are about700Chinese withepilepsy currently, and this condition afflicts at least800000Americans also.Over the past30years, there are about30000drugs were used to carry outepilepsy treatment in animal experiments all over the world, it has achievedgood effect, many epilepsy patients can get relief through the drug therapy,unfortunation, pharmacoyherapy usually fails to achieve long-term remission,it can not inhibit the seizure or recurrent. Currently, the surgery is mainlytaken to treat the refractory epilepsy to resect the epileptic zone, it can notchange the neuronal hyperexcitability and abnormal function of ion channelcompletely, most patients still rely on long-term standard pharmacoyherapy.with the pathogenesis and molecular biology technology developing, the genetherapy as a new method to control and treat epilepsy effectively will bringinnovative and promising alternative.
In the pathogenesis research of epilepsy, it is considered that thehippocampal synaptic reorganization associated with temporal lobe epilepsyclosely, and in hippocampal synaptic reorganization process, moss fibersprouting (MFS) is an important symbol of synaptic reconstruction, and at thesame time with synaptophysin (P38) and synaptic growth-associated protein43(GAP-43) abnormal expressing, this kind of synapse formation reverse toaggratate epilepsy seizures.
The synaptic ultrastructural study found that the morphology, quantity,structure, and function of new synapses in status epilepticus, are different from normal brain neural, these new synapse formation is excitatory circuitry orinexcitatory circuitry, also have aroused wide public attention. The study ofsynaptic ultrastructural shows that most of these recurrent synaptic isasymmetry synapses, and this is a typical characteristics of excitabilitysynaptic. So moss fiber sprouting and granular cell formation of ectopicsynapse is considered to be the important basic structure induced to seizures.
The study of pharmacoyherapy in epilepsy over many years,neuropeptide Y (NPY) as a neurotransmitter and neuromodulator wide spreadover the central nervous system and peripheral nervous system, and it has thehighest concentration in the hippocampus of central nervous system. NPY andits receptors have the relationship with the epileptogenesis and moss fibersprouting, and exogenous NPY has antiepileptic effect. But the neural functionof NPY is very complex in epilepsy model, it appear inconsistent and evenconflicting experimental results with different animal model or differentmethod of administration.
In this study, NPY gene carried by the recombinant adeno-associatedvirus as a vector, is transfered to specific target area of brain, the influence ofNPY gene to epileptic seizure rats which induced by Kainic acid (KA) wasobserved about the MFS, synaptic element expression, synaptic growth-associated protein (GAP-43) in hippocampal CA3using Timm dyeing, PCR,western blot method, and the influence of rAAV2/1-NPY-EGFP onhippocampal synaptic ultrastructural using transmission electron microscope.
Part1The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on seizure and mossy fiber sprouting inhippocampus in rat model induced by kainic acid
Objective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on seizure and mossy fibersprouting in hippocampus in rat.
Methods:30clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=12), KA group (n=12) and controlgroup(n=6). NPY group in which10μl of rAAV2/1-NPY-EGFP (titer5× 1011v.g./ml) were injected to ventricle, KA group, in which amount saline wasinjected to ventricle. Ten minutes later, KA were injected respectively tohippocampal CA3area of two groups, The control group respectively wereinjected in ventricle and CA3area of same amount of saline for two times.Theseizure situation, EEG wave frequency and amplitude were observed afterinjection of2and4week. Mossy fiber sprouting(MFS) in the hippocampuswere detected by Timm’s staining after injection of4w.
Result:
1The seizure degree in rats of NPY group gradually reduced withobservation time. At4W post-injection, in rats of NPY group, the onset ofsymptoms and EEG epileptic discharge frequency and amplitude decreased(P<0.05), compared to rats of positive control group.
2The scores of mossy fiber sprouting in the hippocampal CA3area werehigher in KA group compareed to NPY group.3The control group had noseizure and no significant difference of mossy fiber sprouting.
Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the hippocampal MFS, which provide strong support to the NPYgene therapy for clinical refractory epilepsy.
Part2The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on expression of synaptophysin inhippocampus in rat model induced by kainic acid
Objective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on expression of synaptophysin inhippocampus in rat.
Methods: Clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=24), KA group (n=24) and controlgroup(n=24). NPY group in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle, KA group, in which amountsaline was injected to ventricle. Ten minutes later, KA were injectedrespectively to hippocampal CA3area of two groups, The control grouprespectively were injected in ventricle and CA3area of same amount of saline for two times. Three groups were taken the hippocampal tissue, to observedthe changes of expression of synaptophysin after injection of2and4weekwhich experimentation was performed by fluorescent quantitative PCR andwesten-blot.
Result:
1The seizure degree in rats of NPY groups decreased significantly after4W injection compared with KA groups (P<0.05).
2The results of PCR and Westen-blot on synaptophysin (P38)expression showed that there are obvious difference between NPY groups andKA groups after4W(P <0.05), and there are no difference after2W.
Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the synaptophysin (P38) expression, thus to inhibit epilepsy.
Part3The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on expression of synaptic growth-associatedprotein43(GAP-43) in hippocampus in rat model induced by kainic acid
Objective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on expression of synapticgrowth-associated protein43(GAP-43) in hippocampus in rat.
Methods: Clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=48), KA group (n=48) and controlgroup(n=48). NPY group in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle, KA group, in which amountsaline was injected to ventricle. Ten minutes later, KA were injectedrespectively to hippocampal CA3area of two groups, The control grouprespectively were injected in ventricle and CA3area of same amount of salinefor two times. Three groups were taken the hippocampal tissue, to observedthe changes of expression of GAP-43after injection of2and4week whichexperimentation was performed by immunohistochemical and fluorescentquantitative PCR.
Result: The results of immunohistochemical and fluorescent quantitativePCR on GAP-43expression showed that there are obvious difference between NPY groups and KA groups after4W(P<0.05), and there are no differenceafter2W.
Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the GAP-43expression, thus to inhibit the synaptic reorganization.
Part4The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on ultrastructure features of MFS inhippocampus in rat model induced by kainic acid
Objective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on ultrastructure features of MFSin hippocampus in rat.
Methods: Clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=24), KA group (n=24) and controlgroup(n=24). NPY group in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle, KA group, in which amountsaline was injected to ventricle. Ten minutes later, KA were injectedrespectively to hippocampal CA3area of two groups, The control grouprespectively were injected in ventricle and CA3area of same amount of salinefor two times. Three groups were taken the hippocampal tissue, mossy fibersprouting(MFS) in the hippocampus were detected by Timmhistochemistry,to observed the changes of the synaptic quantity, synapticvesicles density, and the synaptic cleft under the electron microscope.
Result:
1Most of the mossy fiber sprouting synaptic terminals were asym-metric synapses, the quantity of asymmetric synapses increased in KA groupand NPY group after2W, the quantity of asymmetric synapses decreased inNPY group after4W, there are obvious difference between NPY groups andKA groups after4W(P <0.05).
2The synaptic cleft increased in KA group and NPY group after2W, anddecreased in NPY group after4W, there are obvious difference between twogroups after4W(P <0.05).
3It is showed that the quantity of synaptic vesicles reducing, shape of synaptic vesicles being irregular, and the density of synaptic vesicles beingreorganization in KA group and NPY group after2W. The synaptic vesiclesdensity(SVD) is normal after4W, there are obvious difference between NPYgroups and KA groups after4W(P <0.05).
4It is showed that the nucleus swelling, entoblast disappearing, mitoch-ondrial vacuolar degeneration, mitochondrial ridge and membrane dissolved,and the rough endoplasmic reticulum degranulation under the electronmicroscope in KA group and NPY group after2W, comparing to NPY groupafter4W, there are significant difference(P <0.05).
Conclusion: Most of the mossy fiber sprouting synaptics terminals areasymmetric synapses, this kind of new synaptic is irritability, and can beinhibited by NPY. The apoptosis of neuron in hippocampal increased in rat ofepilepsy, and this apoptosis of neuron can be inhibited by NPY.
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