【作者】 沈逸；

【导师】 陈宜张； 罗建红；

【作者基本信息】 浙江大学 ， 生理学， 2008， 博士

【摘要】 白细胞介素-2（Interleukin-2,IL-2）是目前研究比较广泛的细胞因子之一,IL-2不仅具有与T细胞相关的多种免疫调节功能,同时也是一种具有多种生物学特性的细胞因子。除了IL-2的免疫活性之外,神经生物学和神经内分泌学的研究数据表明IL-2在中枢神经系统（central nervous systems,CNS）中发挥重要的作用。除了外周IL-2和它的特异性受体IL-2受体（IL-2R）外,IL-2/IL-2R蛋白分子还广泛存在于额叶皮层、纹状体、海马、下丘脑、蓝斑、小脑、垂体、胼胝体等脑区,也即在CNS中存在着IL-2作用的结构基础,推测可能来源于内源性神经元和胶质细胞的合成和分泌。脑源性的IL-2很可能在其介导的神经调节功能或者CNS紊乱等方面发挥重要的作用;同时,外周的IL-2也能通过转运机制穿过血脑屏障（blood-brainbarrier,BBB）进入一些脑区发挥作用,提示该细胞因子为免疫、内分泌和CNS之间的相互作用提供了体液调节的可能性。然而IL-2的临床应用也引起了许多CNS的副作用,例如IL-2用于抗肿瘤的免疫治疗过程中经常会伴有毒性并发症以及中度或严重的神经精神紊乱,且有认知功能的损伤;在啮齿类动物中,IL-2能引起生物和行为上的变化,并且与一些神经退行性疾病的病理异常相关,例如帕金森病和阿尔茨海默病;IL-2能有效地抑制海马长时程增强（long term potentiation,LTP）的诱导以及维持,提示IL-2可能与学习记忆功能相关。海马是一个富含脑源性IL-2以及IL-2R的脑区并且与学习记忆功能密切相关,因此IL-2在神经精神方面的副作用——认知缺损和记忆丧失等极有可能是IL-2直接作用于该脑区引起的。LTP与长时程抑制（long term depression,LTD）是突触可塑性的两种表现形式（方向相反）,二者的协调表达是学习与记忆能够正常进行的基础。谷氨酸是海马内主要的兴奋性神经递质,它通过与突触后谷氨酸受体结合,激活受体,引发突触兴奋传递。离子型谷氨酸受体作为主要的兴奋性受体参与了神经系统重要功能的调节作用,尤其在突触可塑性的研究中非常关键。离子型谷氨酸受体包括N-甲基-D-天（门）冬氨酸（N-methyl-D-aspartate,NMDA）受体,α-氨基-3-羟-5甲基—异恶唑丙酸（a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate,AMPA）受体以及海藻酸（kinate,KA）受体,前两者主要分布在突触后,而KA受体在突触前与突触后均有分布。AMPA和NMDA受体在海马的突触可塑性的研究中有关键作用。已经证实NMDA受体参与了多种神经系统疾病的病理过程,包括癫痫症,缺血性脑损伤,可能在神经退行性疾病包括帕金森病和阿尔茨海默病中发挥作用,并且NMDA受体掌控着海马LTP和LTD的发生,因此提示我们NMDA受体可能会介入IL-2对突触可塑性的调节作用。有研究发现IL-2能双向调节中脑神经元NMDA受体介导的电流,证实IL-2与中脑神经元NMDA受体之间有着密切的联系。此外,另一种离子型谷氨酸受体——AMPA受体则被认为是突触可塑性变化的重要调节蛋白,通过其反应实现了突触活性的改变。既然离子型谷氨酸受体对海马突触可塑性是非常重要的,而IL-2又介入了海马脑区突触可塑性的调节,因此我们认为IL-2、离子型谷氨酸受体和海马突触可塑性这三者之间很可能存在着某种联系。IL-2是否对海马NMDA受体有作用,或是对于海马的AMPA受体也有作用还不清楚,目前还没有关于IL-2对海马谷氨酸受体作用的相关研究报道。已往关于IL-2与CNS功能的研究主要都是倾向于IL-2是通过与其特异性受体IL-2R结合来发挥生物学效应,但是也有例外。1996年Wang等研究报道IL-2能够直接与阿片受体结合而引起相应的信号转导,说明IL-2并不只是与IL-2R结合后才起作用的。如果IL-2对海马离子型谷氨酸受体有作用,那么是通过什么途径来实现的?是通过与其受体IL-2R结合后引起一系列信号转导起作用或是直接作用于这些受体?这些受体是否具有对IL-2敏感的亚型或靶结构?这些具体的作用机制尚不清楚。此外,研究发现IL-2对多种神经元的生长及存活都有重要的促进作用。IL-2可为大鼠多个脑区包括海马等的原代培养神经元提供营养支持,能促进神经元的形态发育,如神经突的生长等。IL-2在空间学习和记忆相关的脑神经元发育和调节中起到重要的作用。神经元树突是神经元之间信息交换的场所,其长度、直径和分支等各项指标均可反映细胞的功能状态。这些提示我们IL-2对海马神经元树突发育和突触形成可能存在着一定的影响。本学位论文主要通过分子构建、原代神经元培养、电生理技术、活细胞荧光成像及其它相关技术,研究IL-2对离子型谷氨酸受体功能以及培养海马神经元树突发育、突触发生的影响,阐明IL-2的调节作用及其可能机制。第一部分:白细胞介素—2对NMDA受体功能的影响目的:研究IL-2对培养大鼠海马神经元和表达重组NMDA受体的HEK（humanembryonic kidney）293细胞中NMDA受体功能的影响以及初步作用机制。方法:本实验利用全细胞膜片钳技术在体外培养第12天（days in vitro,DⅣ12）到DⅣ14的原代海马神经元以及表达重组NMDA受体的HEK293细胞中通过Y管系统给予NMDA受体激动剂记录NMDA受体介导的全细胞电流(I_NMDA),并用Y管系统给予不同浓度的IL-2处理;同时用逆转录聚合酶链式反应（reversetranscription-polymerase chain reaction,RT-PCR）检测了HEK293细胞中IL-2Rβ亚基的表达情况。结果:我们发现1)在DⅣ12的神经元中,IL-2与NMDA受体激动剂同时作用时,0.01-1 ng/ml IL-2能剂量依赖性快速地抑制I_NMDA峰值,0.1 ng/mlIL-2引起的抑制程度为47±3%（P＜0.001）,而单独Y管给1L-2不能引起细胞的任何反应。2)IL-2对I_NMDA的抑制作用是一种竞争性的抑制作用,NMDA浓度越高,IL-2对I_NMDA的抑制程度就越低;IL-2的存在显著增加了NMDA的EC₅₀值,从42±7μM增加到79±4μM。3)IL-2不改变NMDA受体的反转电位和电流—电压（Ⅰ-Ⅴ）曲线。4)RT-PCR结果显示HEK293细胞中无功能性的IL-2R的存在;在分别表达重组的NMDA受体亚型NR1/NR2A和NR1/NR2B的HEK293细胞中,IL-2也能显著地抑制含有NR2A亚基和NR2B亚基的NMDA受体的电流(I_NR2AandI_NR2B)峰值,并且抑制效率有亚型差异性,分别为54±5%（P＜0.001）和30±4%（P＜0.001）。5)在培养海马神经元中,用10μM ifenprodil阻断NR2B亚型成分电流I_NR2B,也能观察到IL-2对I_NR2A的抑制作用,与HEK293细胞中的结果相似。结论:本实验证实了生理浓度下的IL-2（0.01-1 ng/ml）对培养大鼠海马神经元的NMDA受体有显著的抑制作用,并且这个抑制作用是IL-2浓度依赖的。在表达不同NMDA受体亚基的HEK293细胞中记录两种不同亚型的NMDA受体电流I_NR2A和I_NR2B,IL-2对两者都有抑制作用,并且对I_NR2A的抑制程度大于I_NR2B;IL-2对I_NR2A峰值的抑制作用在培养的海马神经元中也类似。这些结果提示着IL-2可能是直接作用于NMDA受体,而不是IL-2R依赖性的,并且这种抑制作用很可能有NMDA受体亚型的差异性。第二部分:白细胞介素—2对培养大鼠海马神经元的AMPA受体功能的影响目的:研究IL-2对培养大鼠海马神经元AMPA受体功能的影响。方法:仍通过Y管系统给予AMPA受体激动剂以及不同浓度的IL-2,用全细胞膜片钳技术在培养DⅣ12到DⅣ14的大鼠海马神经元中记录AMPA受体介导的全细胞电流(I_AMPA)和AMPA受体介导的微小兴奋性突触后电流（AMPA-mEPSCs）。结果:我们发现1)IL-2与AMPA受体激动剂同时作用时,0.01-1 ng/ml IL-2能剂量依赖性快速地抑制I_AMPA峰值,0.1 ng/ml IL-2引起的抑制程度为55±6%（P＜0.001）,而单独Y管给IL-2也不能引起细胞的任何反应。2)IL-2对I_AMPA的抑制作用是一种竞争性的抑制作用,谷氨酸浓度越高,IL-2对I_AMPA的抑制程度就越小;IL-2增加了谷氨酸的EC₅₀值,从56±8μM增加到144±7μM。3)IL-2显著抑制AMPA-mEPSCs的幅度,抑制程度为24±6%（P＜0.05）,而对频率和衰减时间没有影响。结论:本实验证实了生理浓度下的IL-2（0.01-1 ng/ml）对培养大鼠海马神经元的AMPA受体也有显著的抑制作用,与对NMDA受体的作用类似,但对I_AMPA峰值的抑制程度更为显著;同时IL-2对AMPA-mEPSCs的抑制作用表明IL-2能抑制AMPA受体介导的兴奋性突触的活性,提示AMPA受体可能也介入了IL-2对突触可塑性的调节。第三部分:白细胞介素—2对培养海马神经元树突发育和突触发生的影响目的:观察IL-2对培养海马神经元树突发育和突触发生的影响。方法:本实验在DⅣ5的原代海马神经元中转染能定位在膜上的绿荧光蛋白（Green fluorescentprotein,GFP）F-GFP,用活细胞成像法于第7天观察树突丝的密度、长度和运动情况;于第7、10、14天分别观察树突分支的生长情况;第14天观察树突棘的密度,研究不同作用浓度和时间的IL-2对以上这些形态学特征的影响。用单因素方差分析和双尾t检验对实验结果作显著性检验。结果:我们发现1)IL-2处理后能明显促进培养第7、10、14天神经元树突树的分支发育和长度,增强效应与IL-2作用浓度和时间正相关;10 ng/ml IL-2处理48小时后DⅣ7、DⅣ10、DⅣ14的神经元树突树的分支数目分别增加了48.21±10.74%（P＜0.001）,17.79±5.64%（P＜0.01）和15.28±4.89%（P＜0.05）;而树突树总长度分别增加了43.01±9.43%（P＜0.001）,35.04±7.01%（P＜0.05）和24.48±5.83%（P＜0.01）。这个增强作用也与作用时间正相关。无论是树突树的分支数还是长度,IL-2对DⅣ7神经元的作用最显著。2)10 ng/ml IL-2处理48小时后显著增加了DⅣ7神经元树突丝的运动性,以运动的树突丝的密度（个/100μm）以及运动的树突丝比上树突丝总个数的百分比来表示,分别增加了30.93±12.83%（P＜0.001）和44.18±7.81%（P＜0.001）。3)在DⅣ5到DⅣ7、DⅣ5到DⅣ14以及DⅣ12到DⅣ14这三个时间段分别用10 ng/ml IL-2处理神经元,在DⅣ14天观察,发现IL-2能促进前两组神经元树突棘的密度,分别增加了18.48±6.22%（P＜0.05）和20.57±10.89%（P＜0.01）,但对后一组没有影响（P＞0.05）。结论:这些研究结果提示IL-2能有效促进培养海马神经元树突的发育和突触的形成,尤其是在早期发育阶段更为有效。更多还原

【Abstract】 Interleukin-2（IL-2）is one of the most actively studied cytokines,and it has multiple immunoregulatory functions and biological properties not only related to T-cells.In addition to its immune activities,neurobiological and neuroendocrine data indicated that IL-2 plays roles in the central nervous systems（CNS）.IL-2 and IL-2 receptors（IL-2R） are widely distributed in the brain regions,including frontal cortex,striatum, hippocampus,hypothalamus,locus coeruleus,cerebellum,pituitary and callose,and are potentially produced by neurons and astrocytes;Besides,it has been reported that IL-2 is able to cross the blood-brain barrier（BBB）by saturable and nonsaturable transport mechanisms,which makes it a likely candidate for humoral communication between the immune,endocrine and CNS,suggesting that IL-2 could act as a neuroregulatory factor.IL-2 may prove of therapeutic value in a wide range of pathological conditions,but the significant anticancer effects obtained with IL-2 immunotherapy are often associated with side effects in the CNS,like toxic complications,moderate or severe neurological and mental disorders as well as cognitive impairements.In rodents,exogenous treatment of IL-2 could induce biological and behavioral alterations related to pathogenesis of some degenerative diseases,including Parkinson’s disease and Alzheimer’s disease.It has reported that IL-2 suppresses the induction and maintenance of long-term potentiation（LTP）,a long-lasting increase in synaptic transmission,suggesting that IL-2 may be a modulatory factor in synaptic plasticity.Hippocampus is a region enriched of brain-derived IL-2 and IL-2R proteins,and also closed associated with learning and memory.Thus,side effects in nervous system like cognitive impairments and memory loss induced by IL-2 may be related to direct effects of IL-2 on hippocampus.It is well known that LTP and long term depression（LTD）are the two forms of neuronal plasticity, and are thought to be the substrate of learning and memory.Glutamatic acid is the main excitatory neurotransmitter in hippcampus,and it can initiate synaptic excitatory transmission through binding to and then activating postsynaptic glutamate receptors, which play important roles in regulation of nervous system as major excitatory receptors, especially in synaptic plasticity.There are two major postsynaptic ionotropic glutamate receptor subtypes,that is,N-methyl-D-aspartate（NMDA）receptors andα-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate（AMPA）receptors,which are crucial for mediating hippocampal synaptic plasticity.Activation of NMDA receptors within the CNS represents a major signal for persistent alterations in glutamatergic signaling,like LTP and LTD,which may underlie higher order processes such as learning and memory in the hippocampus and are involved in pathogenesis of several types of neurological disorders such as epilepsy,ischemia-induced brain injury,and neurodegenerative disorders.These studies have implied that NMDA receptors may be a probable target of IL-2 in the regulation of synaptic plasticity and learning and memory. A previous study showed that IL-2 can modulate NMDA receptor-mediated currents (I_NMDA)in native mesolimbic neurons.However,the specific molecular targets of IL-2 on NMDA receptors remain unknown.In addition,AMPA receptors are also important to regulate alterations in synaptic plasticity and their activities are closely linked to the enhancement of synaptic transmission that occurs during LTP.Since ionotropic glutamate receptors and IL-2 are closely related to hippocampal synaptic plasticity,it is possible that there is relationship among IL-2,ionotropic glutamate receptors and synaptic plasticity.NMDA receptors and AMPA receptors may contribute to the possible effects of IL-2 on synaptic plasticity and learning and memory.However,there is no related report at present,we hypothesized that IL-2 may modulate excitability through NMDA receptors and/or AMPA receptors in the hippocampus.Furthermore,it is known that the biological effects of IL-2 are generally exerted through IL-2R.However,not all its effects are initiated by combination with its receptors, for instance,IL-2 can bind toδ-opioid receptors.So,IL-2 may also directly interact with either NMDA receptors or AMPA receptors.It was of interest to study the effects of IL-2, at physiologically relevant concentrations,on these two kinds of receptors.Moreover,IL-2 plays a role in development and regulation of brain neurons.IL-2 can promote development and survival of neurons from various brain regions.For example, it has multiple effects on morphology of hippocampal neurons,including significantly promoting the elongation and branching of neurites in both normal and damaged primary cultures.It is considered that dendrite is the important structure for receiving input signal and the location for integrating and exchanging synaptic information,and the length, diameter and arborazation of dendrites are all crucial to reflect situation of a neuron.It has been reported that many nutrients and stresses affected the formation and function of the whole neuronal circuit by interfering the developmental progress of dendrites.The studies suggested that IL-2 may have impact on dendritic development and synaptogenesis of cultured hippocampal neurons.Therefore,in order to understand effects of IL-2 on NMDA receptors and AMPA receptors as well as dendritic development and synaptogenesis of hippocampal neurons, we performed the following studies by molecular construction,primary neuronal cultures, electrophysiological technique and living cell images.PartⅠ:Interleukin-2 inhibits NMDA receptor-mediated currents directly and may differentially affect subtypesUsing whole-cell patch-clamp recordings,this study investigated the effects of interleukin-2（IL-2）on N-methyl-D-aspartate（NMDA）receptor-mediated currents (I_NMDA)in rat cultured hippocampal neurons and human embryonic kidney（HEK）293 cells expressing recombinant NMDA receptors.We found that IL-2（0.01-1 ng/ml） immediately and significantly decreased peak I_NMDAin cultured neurons,causing a 47±3%inhibition at 0.1 ng/ml（p＜0.001）.The effect of IL-2 on peak I_NMDAamplitude was depended on NMDA concentrations,and the value of EC₅₀for NMDA in the presence of IL-2 was 79±4μM（n=7）,markedly greater than 42±7μM in controls.Interestingly, the peak I_NMDAinduced in HEK 293 cells was also inhibited by IL-2.We also found that IL-2 differentially decreased the peak amplitudes of NR2A- and NR2B-containing NMDA receptor-mediated currents(I_NR2Aand I_NR2B)by 54±5%（P＜0.001）and 30±4% （P＜0.001）,respectively.These results provide new evidence that IL-2 induces rapid inhibition of peak currents of NMDA receptor-mediated responses with possible NR1/NR2A and NR1/NR2B subtype-differentiation,and suggest that the inhibition is possibly mediated by direct interaction between IL-2 and NMDA receptors. PartⅡ:Interleukin-2 inhibits AMPA receptor function in cultured hippocampal neurons of ratsIL-2 is an important neuroregulatory molecule in the CNS.The present study investigated the effects of IL-2 onα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid（AMPA）receptors in cultured hippocampal neurons of rats using whole cell patch-clamp analysis.We found that IL-2（0.01-1 ng/ml）immediately and significantly decreased peak I_AMPAin cultured hippocampal neurons in a concentration-dependent manner,causing a 55±6%inhibition at 0.1 ng/ml（p＜0.001）,similar to the effect on NMDA receptors.The effect of IL-2 on peak amplitude of I_AMPAwas also depended on glutamatic acid concentrations,and the value of EC₅₀for glutamatic acid in the presence of IL-2 was 144±7μM,markedly greater than 56±8μM in controls（n=6）.In addition,we also found that the amplitudes of AMPA miniature excitatory postsynaptic currents（mEPSCs）were significant decreased by 24±6%（p＜0.05）after 0.1 ng/ml IL-2 application,whereas their frequency and decay times were not altered.These results demonstrate that IL-2 induces rapid inhibition of AMPA receptor-mediated responses, indicating that AMPA receptors may be involved in modulation of IL-2 in excitatory synaptic activity in cultured rat hippocampal neurons.PartⅢ:Effects of interleukin-2 on dendritic development and synaptogenesis of cultured hippocampai neurons of ratsThis study has investigated the effects of IL-2 on dendritic filopodia,dendritic arborization,and spine maturation during development in cultured hippocampal neurons of rats.The cultured hippocampal neurons were transfected with F-GFP on days in vitro 5（DIV5）to display the subtle structures of dendrites,and then were treated with IL-2 at various concentrations,respectively,for different time before observation of living cell images.We found that both the dendritic arborization and the length of dendrites per neuron at DIV7,DIV10 and DIV14 were increased by IL-2 treatment in a dose-dependent manner.After treatment for 48 hours,10 ng/ml IL-2 caused 48.21±10.74%（P＜0.001）,17.79±5.64%（P＜0.01）and 15.28±4.89%（P＜0.05）increases in neurons at DIV7,DIV10 and DIV14,respectively;while for the total length of branches, causing 43.01±9.43%（P＜0.001）,35.04±7.01%（P＜0.05）and 24.48±5.83%（P＜0.01）increase at DIV7,DIV10 and DIV14,respectively.The most strongest effects on both dendritic number and length were observed in neurons at DIV7.And there was also a significant increase in the mobility of dendritic filopodia in the neurons at DIV7 treated with 10 ng/ml IL-2 for 48 h from DIV5 to DIV7,causing a 30.93±12.83%increase in mobile filopodia/100μm（P＜0.001）and 44.18±7.81%increase in ration of mobile/total filopodia（P＜0.001）,but no significant change was observed in filopodia density and length.In addition,IL-2 also caused an increase in the spine density of neurons at DIV14 either treated with IL-2 from DIV5 to DIV7 or from DIV5 to DIV14,causing 18.48±6.22%increase in the former group（P＜0.05）and 20.57±10.89%increase in the latter group（P＜0.01）,respectively,but did not affect neurons with IL-2 treatment from DIV12 to DIV14.These results indicate that IL-2 may affect the dendritic development and formation of synapses of cultured hippocampal neurons,especially during the early developmental stage of neurons.更多还原