【作者】 王丽红；

【导师】 王怀经； 李振中； Mark S. Kindy； 邢子英；

【作者基本信息】 山东大学 ， 神经生物学， 2009， 博士

【摘要】 神经肽免疫反应(neuropeptide-immunoreactive,NP-IR)神经元和神经丝免疫反应(neurofilament-immunoreactive,NF-IR)神经元是背根神经节(dorsal rootganglion,DRG)神经元的两个主要表型。NP-IR神经元周围突属于无髓或薄髓纤维,分布于内脏和皮肤,可以合成并释放P物质(substance P,SP)等生物活性物质。NF-IR神经元周围突则属于有髓纤维,分布于肌梭,主要参与牵张反射刺激,与本体感觉的信息传递有关。SP是DRG神经元内的神经递质,在神经元的胞体内合成,经轴突运输至神经末梢,在炎症、应激等刺激下释放,发挥生物学效应。神经丝是神经元细胞骨架结构的一个重要组成部分,参与维持神经元正常的形态结构。神经丝主要有轻(NF-L)、中(NF-M)、重(NF-H)三种亚型,其分子量分别为68 kDa,160 kDa和200 kDa。神经丝-200(neurofilament 200,NF-200)是构成轴突的主要骨架结构,在DRG神经元发育过程中有表达,是NF-IR神经元表型的一个重要指标。单涎性神经节苷脂(monosiaganglioside,GM1)在神经元的发育、分化和存活等生理状态和病理状态下均具有营养作用。体内与体外实验皆表明,GM1可以发挥神经营养因子样作用。体外实验表明,神经元诱导分化过程中伴随着GM1生物合成的改变和核膜GM1含量的增高。神经营养因子(neurotrophins,NTs)在周围神经系统的发育和存活中具有重要的调节作用。神经生长因子(nerve growth factor,NGF)、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)和神经营养素-3(neurotrophin-3,NT-3)都是NTs家族的成员。研究表明,外源性应用这些营养因子可以保护受损神经元,刺激轴突再生。NGF在感觉神经元的发育过程中,对于初级传入神经元的存活和表型的形成具有重要的影响作用。体外培养成年DRG感觉神经元发现,部分DRG感觉神经元的神经肽表型是固有的,并且其表型的表达程度可以由NGF来调节。在DRG神经元受损时,NGF还可以逆转具有高亲和力NGF受体的DRG神经元中神经丝mRNA的减少。BDNF在中枢和周围神经系统的发育过程中起着重要的作用,可以调节神经元的存活、生长和分化。在DRG感觉神经元中,BDNF主要存在于小型和中型亚群DRG感觉神经元,可以顺向转运至神经元的周围突和中枢突。体内实验表明,BDNF的存在对于DRG感觉神经元的存活和功能的维持具有非常重要的作用。体外实验也表明,部分感觉神经元的存活依赖于BDNF的存在。NT-3可作为促细胞分裂因子或诱导神经元分化的因子,维持胚胎发育中神经元的存活,并促进其分化与增殖。NT-3在感觉神经元和交感神经元的存活和分化,以及神经突起发生、突触形成和轴突生长中均具有非常重要的作用。研究发现,DRG感觉神经元可能是脊髓神经营养因子如NT-3的来源之一,并且在脊髓损伤后DRG感觉神经元内的NT-3可以顺行转运至脊髓,有利于脊髓神经元的再生。靶组织在维持神经元的功能和神经-肌间的信息传递中,发挥着重要作用。运动神经元与其支配的骨骼肌(skeletal muscle,SKM)之间具有密切的相互依赖关系。两种组织之间的重要信息传递是由神经-肌接头(neuromuscular junction,NMJ)介导的。在这两种组织的其它部位释放和接收各种因子也是它们之间相互影响的一种方式。神经.肌之间的信息传递之一是神经营养因子和其他分子的相互交换。体外实验发现,新生哺乳动物NF-IR阳性DRG神经元,需要SKM提取物中的一种神经营养因子来维持其存活。实验表明,在神经元的发育过程中,靶源性神经营养因子对于支配肌细胞的不同神经元的存活和分化具有重要的生物学作用。感觉神经末梢可以为去神经支配肌组织提供神经营养因子,SKM的肌梭也受DRG感觉神经元支配。GM1、NGF、BDNF、NT-3和靶组织SKM对维持神经元的功能都发挥重要的作用。然而,GM1、NGF、BDNF、NT-3单独应用或与靶组织SKM联合应用是否影响DRG感觉神经元的表型目前尚不清楚。基于以上研究背景,本研究在体外建立DRG神经元单纯培养和DRG神经元与SKM细胞联合培养的细胞模型,通过检测体外培养的DRG神经元中SP-IR和NF-200-1R神经元的表达情况,来分析GM1、NGF、BDNF、NT-3和SKM细胞单独应用以及GM1、NGF、BDNF、NT-3分别与SKM细胞联合应用对DRG神经元表型的影响作用,为初级传入神经元表型变化的发育神经生物学提供新的理论和实验依据。第一部分GM1和骨骼肌对培养的IBRG神经元表型的影响作用胚胎第12.5天(embryonic day 12.5,E12.5)和胚胎第19.5天(embryonic day19.5,E19.5)的大鼠DRG神经元,分别进行体外单纯分散培养以及与SKM细胞的联合培养,体外培养2 d后,分为8组。(1)E12.5对照组:E12.5DRG神经元单纯培养,不施加任何干预因素,继续在培养液内培养4 d;(2)E19.5对照组:E19.5DRG神经元单纯培养,不施加任何干预因素,继续在培养液内培养4 d;(3)E12.5GM1组:E12.5DRG神经元单纯培养,用GM1(终浓度10μg/ml)孵育4 d;(4)E19.5GM1组:E19.5DRG神经元单纯培养,用GM1(终浓度10μg/ml)孵育4 d:(5)E12.5SKM组:E12.5DRG神经元与SKM细胞联合培养,不施加任何干预因素,继续培养4 d;(6)E19.5SKM组:E19.5DRG神经元与SKM细胞联合培养,不施加任何干预因素,继续培养4 d;(7)E12.5GM1+SKM组:E12.5DRG神经元与SKM细胞联合培养,用GM1(终浓度10μg/ml)孵育4 d:(8)E19.5GM1+SKM组:E19.5DRG神经元与SKM细胞联合培养,用GM1(终浓度10μg/ml)孵育4 d。在培养过程中,用倒置相差显微镜动态观察各组标本在不同时间的生长状态。终止培养后,用扫描电子显微镜观察单纯培养中DRG神经元胞体、突起及神经末梢的形态以及联合培养中DRG神经元胞体、突起的形态以及神经末梢与SKM之间的关系;用免疫荧光双标技术来检测单纯培养和联合培养中DRG神经元中SP-IR和NF-200-IR神经元的表达变化。实验结果如下:(1)倒置相差显微镜和扫描电子显微镜观察发现,DRG神经元的突起跨过SKM细胞或者到达并分布于SKM细胞表面,在SKM细胞自发性收缩的牵动下,DRG神经元的突起也随之移动。(2)GM1可以促进体外分散培养的E12.5DRG神经元中SP和NF-200的表达(P＜0.05),而不能促进体外分散培养的E19.5DRG神经元中SP和NF-200的表达。(3)靶组织SKM细胞可以促进体外培养的E12.5 DRG神经元中NF-200的表达(P＜0.05),而不能促进SP的表达;靶组织SKM细胞对体外培养的E19.5DRG神经元中SP和NF-200的表达无影响作用。(4)GM1和靶组织SKM细胞对促进E12.5DRG神经元SP和NF-200的表达具有协同作用(P＜0.001);而GM1和靶组织SKM细胞对E19.5DRG神经元SP和NF-200的表达无影响作用。以上结果表明:在体外合适的培养环境条件下,DRG神经元和SKM细胞可在形态上建立神经-肌连接;在到达靶组织SKM之前(E12.5)的DRG神经元NF-IR表型依赖于靶组织SKM的存在,而NP-IR表型并不依赖于靶组织SKM的存在,这表明NP-IR表型是由其内在固有的特性所决定的;GM1可以促进E12.5 DRG神经元SP和NF-200的表达,这表明在到达靶组织SKM之前(E12.5)的DRG神经元表型具有可塑性;GM1和靶组织SKM细胞促进E12.5DRG神经元SP和NF-200表达的协同作用进一步表明在到达靶组织SKM之前(E12.5)的DRG神经元表型具有明显的可塑性;在到达靶组织SKM之后(E19.5)的DRG神经元表型并不依赖于靶组织SKM或GM1的存在,这表明DRG神经元表型在这一阶段的可塑性变化不明显。这些结果为理解初级传入神经元表型变化的规律提供了新的理论基础和实验依据,并为进一步深入了解GM1和靶组织SKM对神经元表型在发育过程中变化的影响作用,提供了新的思路和方法。第二部分神经营养因子和骨骼肌对培养的DRG神经元表型的影响作用体外培养的DRG神经元取自Wistar大鼠第12.5 d的胚胎。无菌条件下进行体外DRG神经元单纯培养以及与SKM细胞的体外联合培养,体外培养2 d后,分为8组。(1)对照组:单纯培养的胎鼠DRG神经元,不施加任何干预因素,继续在培养液内培养4 d;(2)NGF组:单纯培养的胎鼠DRG神经元,用NGF(10 ng/ml)孵育4 d;(3)BDNF组:单纯培养的胎鼠DRG神经元,用BDNF(10 ng/ml)孵育4 d;(4)NT-3组:单纯培养的胎鼠DRG神经元,用NT-3(10 ng/ml)孵育4 d;(5)SKM组:胎鼠DRG神经元与SKM细胞联合培养,不施加任何干预因素,继续在培养液内培养4 d;(6)SKM+NGF组:胎鼠DRG神经元与SKM细胞联合培养,用NGF(10 ng/ml)孵育4 d;(7)SKM+BDNF组:胎鼠DRG神经元与SKM细胞联合培养,用BDNF(10 ng/ml)孵育4d:(8)SKM+NT-3组:胎鼠DRG神经元与SKM细胞联合培养,用NT-3(10 ng/ml)孵育4 d。在培养过程中,用倒置相差显微镜动态观察各组标本在不同时间的生长状态。终止培养后,用免疫荧光双标技术检测单纯和联合培养中DRG神经元SP-IR和NF-200-IR神经元表达的比例,用Western blot技术检测联合培养标本中SP和NF-200蛋白的表达。实验结果如下:(1)NGF可以促进体外分散培养E12.5DRG神经元SP(P＜0.01)和NF-200(P＜0.05)的表达,并且NGF和靶组织SKM细胞对促进E12.5DRG神经元SP和NF-200的表达具有协同作用(P＜0.01)。(2)BDNF可以促进体外分散培养E12.5DRG神经元SP和NF-200的表达(P＜0.05),并且BDNF和靶组织SKM细胞对促进E12.5DRG神经元NF-200的表达具有协同作用(P＜0.05)。(3)NT-3和靶组织SKM细胞均可以促进体外分散培养E12.5DRG神经元NF-200的表达(P＜0.05),但不能促进神经元SP的表达;并且NT-3和靶组织SKM细胞对促进E12.5DRG神经元NF-200的表达具有协同作用(P＜0.001)。以上结果表明:神经营养因子NGF、BDNF和NT-3均可影响到达靶组织SKM之前(E12.5)的DRG神经元表型的表达变化。但NGF、BDNF和NT-3对DRG神经元表型的影响作用程度不同,NGF和BDNF单独应用时可影响NP-IR和NF-IR两种神经元表型,而NT-3则只能影响NF-IR神经元表型。NGF与靶组织SKM联合应用时,NP-IR和NF-IR两种神经元表型均表达增加,这显示NGF与SKM对两种神经元表型的影响具有协同作用。BDNF、NT-3分别与靶组织SKM联合应用时,仅对NF-IR神经元表型的影响具有协同作用。这表明DRG神经元表型在发育过程中的可塑性变化在不同的环境条件下是不同的,但神经营养因子NGF、BDNF、NT-3与SKM的协同作用机制尚需进一步探讨。这些结果为进一步深入了解各种神经营养因子和靶组织SKM对发育过程中神经元表型变化的影响机制,提供了一条新的研究途径。更多还原

【Abstract】 The neuropeptide-immunoreactive(NP-IR) and neurofilament-IR(NF-IR) neurons are two major phenotypical classes in dorsal root ganglion(DRG).NP-IR neurons are considered to be with unmyelinated or thinly myelinated nociceptive afferents which are considered to innervate skin and viscera.Neuropeptide-IR neurons synthesized and released a variety of bioactive substances,including substance P(SP).Whereas neurofilament-IR neurons typically have myelinated axons which are considered to innervate muscle spindle,involved in the stretch reflex and proprioception.The neurotransmitters of SP are located in the DRG neurons,synthesized in the cell bodies and transported to the axon terminals in the state of inflammation,stress and other stimuli.Neurofilaments are the important structural component of the neurons cytoskeleton,involved in the maintenance of normal structure and morphology.There are three neurofilament subtypes,NF-light, medium,and heavy,that is,NFL,NFM and NFH.The molecular weight was 68 kDa,160 kDa and 200 kDa,respectively.NF-200 is the main component of the cells skeleton structure.In DRG neurons,it is expressed during development.It is an important indicator for the identification of NF-IR neuronal phenotype.Monosialoganglioside(GMI) plays a nutritional role in neuronal development, differentiation,and survival in the condition of physiological and pathological.Both in vivo and in vitro experiments showed that,GM1 can play a neurotrophic factor-like role in neurons.In vitro,the differentiation of neurons is accompanied by the biosynthesis of GM1 and the content increased of membrane GM1.The motor neurons and skeletal muscle fibers they innervate are strongly dependent on each other.Important communication between both tissues is mediated through the neuromuscular junction.However,release and reception of various factors at other parts of both tissues must also be considered as means of mutual influences.Exchange of neurotrophins and other molecules is likely to be an important source of nerve-muscle communication.The in vitro survival of neonatal mammalian neurofilament-positive DRG neurons requires the presence of a neurotrophic factor present in skeletal muscle extract.The experimental results show that during the development of neurons,target-derived neurotrophic factor has an important role in the survival and differentiation of the innervated neurons. Skeletal muscles(i.e.,muscle spindle) are also innervated by DRG sensory neurons which are considered as neurofilament-IR neurons.Neurotrophins(NTs) are the most profound known regulators of survival in the developing peripheral nervous system.Nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF),and neurotrophin 3(NT-3) are three members of the neurotrophic factor(neurotrophin) family.Exogenous administration of these factors has protective properties for injured neurons and stimulates axonal regeneration.NGF has a key role in the survival and establishment of the phenotype of responsive primary afferent neurons during development.Some neuronal phenotypes of neuropeptides are retained and can remain sensitive to NGF regulation in aged DRG neuronal cultures.NGF regulates sensory neuron phenotype by elevated expression of ion channels and receptors contributing to pain. After the DRG neurons damaged,NGF counteracted this injury-induced reduction of neurofilament mRNA but only in neurons with high-affinity NGF receptors. BDNF plays a critical role in the development of the central and peripheral nervous systems,supports proliferation,differentiation and survival of neurons.It has been shown that BDNF is present in a subpopulation of small- to medium-sized DRG sensory neurons and is anterogradely transported in both the peripheral and central processes.The presence of BDNF is necessary for the maintenance of the DRG neurons in vivo.In vitro studies suggest that survival of many populations of cranial sensory neurons initially depends on BDNF.NT-3 is also a key member of neurotrophin family.NT-3 has an important role in survival and differentiation of neurons served as a factor of promoting cell division or inducing neuronal differentiation.NT-3 plays an important role in survival and differentiation of sensory and sympathetic neurons,sprouting of neurites,synaptic reorganization,and axonal growth.GM1,NGF,BDNF,NT-3 and target tissues are essential for the maintenance of the function of neurons.However,much less is known about the influence of GM1, NGF,BDNF,NT-3 and target muscle cells on DRG neuronal phenotypes.And also, association of target muscle cells with GM1,NGF,BDNF and NT-3 on influence of DRG neuronal phenotype remains unknown.Here we have established a single DRG neurons culture model and a neuromuscular co-culture model of DRG neurons and skeletal muscle cells to test what extent to the expression of SP and NF-200 in DRG neurons in both cell culture models.PartⅠEffects of GM1 and skeletal muscle cells on dorsal root ganglion neuronal phenotypes in vitroDRG was harvested at embryonic days 12.5(El2.5) and E19.5,and cultured without and with skeletal muscle cells(SKM).After cultured for 2 d,the cultures were divided into 8 groups.(1) E12.5 control:E12.5 DRG neurons was cultured continuously for another 4 d;(2) E19.5 control:E19.5 DRG neurons was cultured continuously for another 4 d;(3) E12.5 GM1 group:E12.5 DRG neuronal culture was exposed to GM1(final concentration,FC:10μg/ml) for another 4 d;(4) E19.5 GMI group:E19.5 DRG neuronal culture was exposed to GMI(final concentration, FC:10μg/ml) for another 4 d;(5) E12.5 SKM group:The co-culture of E12.5 DRG neurons and SKM cells was cultured for another 4 d;(6) E19.5 SKM group:The co-culture of E19.5 DRG neurons and SKM cells was cultured for another 4 d;(7) E12.5 GMI+SKM group:The co-culture of E12.5 DRG neurons and SKM cells with GMI exposure(FC:10μg/ml) for another 4 d;(8) E19.5 GM1+SKM group: The co-culture of E19.5 DRG neurons and SKM cells with GMI exposure(FC:10μg/ml) for another 4 d.All the cultured living cells were observed under inverted phase contrast microscope.The morphology of DRG neurons and the relationship between DRG neuronal terminals and SKM cells were observed by using scanning electron microscope(SEM) at 6 d of culture age.DRG cultures and neuromuscular co-cultures were processed for double immunofluorescent labeling of MAP2 and SP or NF-200 at 6 d of culture age.The results are as follows:(1) Under inverted phase contrast microscope and scanning electron microscopy,it has shown that many DRG neurons neurites crossed or distributed to the surface of the SKM cells.(2) GM1 could promote SP and NF-200 expression in the cultures of DRG harvested at E12.5(P＜0.05).GMI had not effects on SP and NF-200 expression in the cultures of DRG harvested at E19.5.(3) Target SKM could promote expression of NF-200(P＜0.05) but not SP in the cultures of DRG harvested at E12.5.Target skeletal muscle cells had not effects on SP and NF-200 expression in the cultures of DRG harvested at E19.5.(4) GM1 and target skeletal muscle cells had synergistic effects on the expression of SP and NF-200 in the cultures of DRG harvested at E12.5(P＜0.001). Target skeletal muscle cells combined with GM1 had not effects on SP and NF-200 expression in the cultures of DRG harvested at E19.5.The results indicate that the contact between DRG neurons and skeletal muscle cells was founded in the neuromuscular co-culture of DRG neurons and skeletal muscle cells.These data suggest that neurofilament-IR neurons,but not neuropeptide-IR neurons,are partially dependent on the presence of target skeletal muscle cells.In the present study,GM1 promoted SP and NF-200 expression in E12.5 DRG neuronal cultures.Target skeletal muscle cells only induced NF-200, but not SP expression in E12.5 DRG neuronal cultures.Furthermore,GM1 and target skeletal muscle cells had synergistic effects on SP and NF-200 expression in E12.5 DRG neuronal cultures.These results indicated that the cultured DRG neurons which were harvested at times before(at E12.5) sensory neurons contact peripheral targets in vivo have some plasticity in the presence of GM1.However, the DRG neurons phenotype harvested after(at E19.5) sensory neurons contact peripheral targets in vivo are not dependent on the presence of target skeletal muscle cells and GM1.These results offered new clues for a better understanding of the association ofGMl and skeletal muscle with neuronal phenotypes.PartⅡEffects of neurotrophins and skeletal muscle cells on dorsal root ganglion neuronal phenotypes in vitroDRG cell culture preparations utilized E12.5 d Wistar rats.Under aseptic conditions,DRG neurons are cultured as dissociate cells or cultured as the neuromuscular co-cultures of DRG neurons and SKM cells.After cultured for 2d, the cultures were divided into 8 groups.(1) Control:DRG neurons was cultured continuously for another 4 d;(2) NGF group:DRG neuronal culture was exposed to NGF(FC:10 ng/ml) for another 4 d;(3) BDNF group:DRG neuronal culture was exposed to BDNF(FC:10 ng/ml) for another 4 d;(4) NT-3 group:DRG neuronal culture was exposed to NT-3(FC:10 ng/ml) for another 4 d;(5) SKM group:The co-culture of DRG neurons and SKM cells was cultured for another 4 d;(6) SKM+NGF group:The co-culture of DRG neurons and SKM cells with NGF exposure(FC:10 ng/ml) for another 4 d;(7) SKM+ BDNF group:The co-culture of DRG neurons and SKM cells with BDNF exposure(FC:10 ng/ml) for another 4 d; (8) SKM+ NT-3 group:The co-culture of DRG neurons and SKM cells with NT-3 exposure(FC:10 ng/ml) for another 4 d.All the cultured living cells were observed under inverted phase contrast microscope.At 6 d of culture age,all above cultured samples were processed for detecting the expression of SP and NF-200 in DRG neurons by double immunofluorescent technique.The neuromuscular co-culture samples were processed for detecting the expression of SP and NF-200 in DRG neurons by Western blot analysis.The results are as follows:(1) NGF could promote SP(P＜0.01) and NF-200(P＜0.05) expression in the cultures of DRG harvested at E12.5 d,and NGF and target SKM cells had synergistic effects on the expression of SP and NF-200 in the cultures of DRG harvested before sensory neurons contact peripheral targets in vivo(E12.5).(2) BDNF could promote SP and NF-200 expression in the cultures of DRG harvested at E12.5 d(P＜0.05),and BDNF and target SKM cells had synergistic effects on the expression of NF-200 in the cultures of DRG harvested at E12.5.(3) NT-3 and target SKM cells could promote expression of NF-200(P＜0.05) but not SP in the cultures of DRG harvested at E12.5 d,and NT-3 and target SKM cells had synergistic effects on the expression of NF-200 in the cultures of DRG harvested at E12.5(P＜0.001).The results indicate that NGF,BDNF or NT-3 have an effect on the expression of DRG neurons phenotype in the cultures of DRG harvested before sensory neurons contact peripheral targets in vivo(E12.5).Neurothophins and target tissues have different effects on the phenotype of DRG neurons.NGF and BDNF play a key role in the NP-IR and NF-IR phenotype of DRG neurons,but NT-3 only have a role in the NF-IR phenotype of DRG neurons.NGF and SKM cells had synergistic effects on the two major phenotypicai classes in DRG.BDNF and NT-3 had synergistic effects on the NF-IR phenotype of DRG neurons in the presence of SKM cells.These datas showed that the DRG neurons phenotypic plasticity is different in different conditions during the development.The mechanism involved in these effects should be clarified by further study.These results provided a new way to find the mechanism of the changes of neuronal phenotypes affected by the NTs and target SKM cells.更多还原