细胞因子rhIL—1β,rhIL—2及rhIL—6对大鼠海马神经元营养作用的研究

本工作采用显微测量，图像分析和流式细胞分析方法，研究了细胞因子ｒｈＩＬ－１β，ｒｈＩＬ－２及ｒｈＩＬ－６对新生大鼠海马培养神经元突起生长发育，胞体生长状态，神经元存活数及培养过程中蛋白总量影响，结果显示，这３种细胞因子均对海马培养神经元的突起生长，胞体发育以及发育过程中神经元的蛋白质民具有明显的促进作用，并且在提高海马培养神经元存活，延长存活时间方面有较显著的效应，提示白介素类细胞因子ｒｈＩＬ－１     

青藤碱防护脂多糖所致PD大鼠黑质多巴胺能神经元损伤实验研究

目的观察中药青藤碱(SN)对脂多糖(LPS)所致大鼠黑质多巴胺能(DA)神经元损伤的保护作用。方法黑质内注射LPS制作PD大鼠模型。应用SN对实验动物进行预处理。实验分为对照组、PD组及SN组,采用行为学、酪氨酸羟化酶(TH)、环氧化酶-2(COX-2)等免疫组化及免疫印迹技术等观察SN对LPS所致黑质DA能神经元损伤的保护作用。结果对照组大鼠无任何行为变化,PD组大鼠平均旋转圈数为196.90±9.52,SN组明显减少,为98.79±8.81,差异非常显著(P<0.01)。TH免疫组化表明对照组黑质TH阳性神经元数量较多,胞体较大,突起明显;PD组TH阳性神经元数量明显减少,甚至消失,神经元胞体萎缩,突起亦不清晰,差异非常显著(P<0.01)。SN组TH阳性神经元数量与PD组相比明显增加(P<0.01),神经元形态变化亦不明显。COX-2免疫组化表明对照组黑质致密部偶见COX-2阳性细胞,PD组可见大量散在分布的COX-2阳性细胞,SN组COX-2阳性细胞数与PD组相比明显减少,差异非常显著(P<0.01)。小胶质细胞特异性抗体(OX-42)免疫组化表明对照组大鼠黑质小胶质细胞多呈静止的分枝样状态,PD组多...     

bFGF和EGF对体外培养新生大鼠海马神经元促生长作用的研究

研究不同浓碱性成纤维细胞生长因子和表皮生长因子对新生大鼠海马神经元生长活性的影响。方法取新生大鼠海马组织进行原代体外分离培养，从细胞形态学方面及应用ＭＴＴ法观察ｂＦＧＦ和ＥＧＦ对大鼠海马神经元的影响。结果ｂＦＧＦ能明显促进体外培养大鼠海马神经元存活及突起生长，ＥＧＦ也能促进其存活，尤以促进突起生长更为明显；     

脑衰反应调节蛋白-2（CRMP-2）促进海马神经元轴突生长

目的研究脑衰反应调节蛋白2（collapsin response mediator protein,CRMP-2）对原代海马神经元轴突生长的影响,探讨CRMP-2在神经元轴突生成中的作用。方法每次原代取孕18d SD大鼠1只,每只孕鼠含胎鼠约12~15只。显微镜下分离12~15只胎鼠双侧海马组织,消化法培养原代海马神经元。原代海马神经元培养采用核电转实验转染绿色荧光蛋白（enhanced green fluorescence protein,EGFP）和野生型CRMP-2（wild type（wt）CRMP2）和突变型T514D-CRMP2（突变体,模拟失活型CRMP-2）。培养72h固定做免疫荧光双标,分别绿色荧光蛋白（EGFP）和轴突标志物Tau-1,采用激光共聚焦显微镜和普通光学显微镜观察海马神经元形态变化。结果转染EGFP载体的对照组神经元正常发育,培养至72h,神经元轴突特异性表达标志物蛋白Tau-1;过表达了wtCRMP-2的神经元除了生成一条长的特异性表达Tau-1的轴突,另外一条突起也发育成了特异性表达Tau-1的轴突;而过表达了失活型CRMP-2的神经元发育与正常组相比无差异。结论野生型CRMP-2可明显促进轴突生长,而转染模拟磷酸化CRMP-2的突变体T514D-CRMP2则没有显示任何的促进作用。     

冈田酸抑制神经元轴突生长

目的研究蛋白磷酸酯酶2A（protein phosphatase2A，PP2A）抑制剂冈田酸（okadaic acid，OA）对胎鼠海马神经元轴突生长的影响，探讨PP2A在神经元轴突生成中的作用。方法实验分为二甲基亚砜（Dimethyl sulfoxide，DMSO）对照组和0A处理组，OA处理组依药物浓度又分为5、10、20、30、40nmol／L5个组，激光扫描共聚焦显微镜和普通光学显微镜观察海马神经元形态变化。结果对照组原代海马神经元培养48h后神经元轴突已经形成。当给予不同浓度的0A处理后普通光学显微镜下可见原代海马神经元突起生长均明显受到抑制，突起长度缩短随OA浓度增加抑制作用逐渐增强，对照组平均轴突长度为185pm，而不同浓度OA处理后则分别下降至150／μm、100,am、80ptm、75μm、50μm。结论OA抑制原代海马神经元轴突生长，提示PP2A在神经元轴突生成中起重要作用。     

BDNF,NT-3对体外培养的胚鼠脊髓AChE和NADPH-d阳性神经元生长发育的影响

利用AChE和NADPH-d酶组织化学染色法研究了脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)和神经营养因子-3(neurotrophin-3,NT-3)对离体培养的胚胎大鼠脊髓胆碱能神经元和一氧化氮能神经元生长发育的影响.结果显示:BDNF处理组和NT-3处理组AChE阳性神经元数和NADPH-d阳性神经元数均显著高于对照组(P＜0.05).BDNF组AChE阳性神经元和NADPH-d阳性神经元胞体平均直径、每细胞突起数和最长突起长度均显著高于对照组(P＜0.05).NT-3组NADPH-d阳性神经元的生长发育与对照组无明显差异,仅AChE阳性神经元的每细胞突起数和最长突起长度显著高于对照组(P＜0.05),对胞体发育无影响.结果提示:BDNF,NT-3促进脊髓神经元的存活和生长发育,二者的作用具有选择性和特异性.     

凝血酶对原代培养海马神经元游离钙浓度的影响

目的研究原代培养的海马神经元内游离Ca2 水平及凝血酶的影响.方法大鼠海马神经元进行体外原代培养,用钙离子指示剂Fura-2双波长法测定海马神经元内游离[Ca2 ]i及不同浓度的凝血酶作用后细胞内[Ca2 ]i.结果原代培养的海马神经元生长旺盛,密度高,符合实验要求.在胞外Ca2 浓度为0.0 mmol/L时,静息状态下海马神经元游离[Ca2 ]i为(79.83±18.78)nmol/L.当胞外Ca2 浓度为1.3 mmol/L时,海马神经元游离[Ca2 ]i为(106.41±22.53)nmo1/L.(1～40)U/ml凝血酶可使海马神经元内游离Ca2 水平显著升高,与对照组相比均有显著性差异(P＜0.01).随凝血酶浓度的增加,胞内游离[Ca2 ]i之呈剂量依赖性增加.结论凝血酶可使原代培养的海马神经元内游离Ca2 浓度明显升高.     

白介素-2对海马神经细胞的功能调控

采用形态学、电生理学和生物化学等方法，研究了重组人白介素-2(rhIL-2)对新生大鼠海马培养神经元生长发育、平均蛋白总量、膜电活动以及细胞内游离钙离子浓度的影响。结果显示．rhIL-2对海马培养神经元的突起生长、胞体发育以及发育过程中神经元的蛋白质合成具有明显的促进作用，并且在提高海马培养神经元存活、延长存活时间方面有较为显著的效应。细胞内记录结果显示．rhIL-2可改变海马神经元的膜兴奋性，该作用可被纳洛酮减弱。提示阿片受体可能参与介导rhIL-2的作用过程。另外，采用fura-2荧光探针标记法发现，rhIL-2能够通过胞外钙离子的流入和胞内钙库的释放提高海马神经元胞浆内游离钙离子的浓度。上述研究结果初步表明，细胞因子rhIL-2能够调控海马神经元的功能活动。     

BDNF，NT—3对体外培养的胚鼠脊髓AChE和NADPH—d阳性神经元生长发育的影响

利用AChE和NADPH d酶组织化学染色法研究了脑源性神经营养因子 (brain derivedneurotrophicfac tor ,BDNF)和神经营养因子 3(neurotrophin 3,NT 3)对离体培养的胚胎大鼠脊髓胆碱能神经元和一氧化氮能神经元生长发育的影响。结果显示 :BDNF处理组和NT 3处理组AChE阳性神经元数和NADPH d阳性神经元数均显著高于对照组 (P <0 .0 5 )。BDNF组AChE阳性神经元和NADPH d阳性神经元胞体平均直径、每细胞突起数和最长突起长度均显著高于对照组 (P <0 .0 5 )。NT 3组NADPH d阳性神经元的生长发育与对照组无明显差异 ,仅AChE阳性神经元的每细胞突起数和最长突起长度显著高于对照组 (P <0 .0 5 ) ,对胞体发育无影响。结果提示 :BDNF ,NT 3促进脊髓神经元的存活和生长发育 ,二者的作用具有选择性和特异性。     

Ⅰ型电压门控钠离子通道蛋白在大鼠海马区不同类型神经元上的表达差异

目的 研究Ⅰ型电压门控钠离子通道(Nay1.1)蛋白在大鼠海马区不同类型神经元上的表达情况.方法 采用成年SD大鼠脑组织切片.通过免疫组织化学方法和荧光标记激光共聚焦显微镜法观察比较海马区不同类型神经元上Nay1.1蛋向的表达.结果 在SD大鼠的海马中,Nay1.1蛋白在各区的锥体神经元和齿状回的颗粒细胞胞体上的表达均很微弱.而散在分布的中间神经元上则有很强的阳性表达.结论 SD大鼠海马区散在分布的中间神经元上Nav1.1蛋白的表达较强,而投射神经元CA1-4各区的锥体神经元和齿状回的颗粒细胞则表达微弱:推测不同类型的神经元上可能存在不同亚型的钠离子通道优势分布.     

Neuregulin-1beta induces neurite extension and arborization in cultured hippocampal neurons

Neuregulin-1 (NRG-1) growth and differentiation factors and their erbB receptors are hypothesized to promote embryonic hippocampal neuron differentiation via as yet unknown mechanisms. We have found that NRG-1beta increases the outgrowth of primary neurites, neuronal area, total neurite length, and neuritic branching in E18 hippocampal neurons. NRG-1beta effects on neurite extension and arborization are similar to, but not additive with, those of brain-derived neurotrophic factor and reflect direct NRG-1 action on hippocampal neurons as these cells express the NRG-1 receptors erbB2 and erbB4, the erbB-specific inhibitor PD158780 decreases NRG-1beta induced neurite outgrowth, and NRG-1beta stimulation induces p42/44 ERK phosphorylation. Pharmacological inhibition of p42/44 ERK and protein kinase C (PKC), but not PI3K or p38 MAP kinase, inhibits NRG-1beta-induced neurite extension and elaboration. We conclude that NRG-1beta stimulates hippocampal neurite extension and arborization via a signaling pathway that involves erbB membrane tyrosine kinases (erbB2 and/or erbB4), p42/44 ERK, and PKC.     

Neuregulin-1β induces neurite extension and arborization in cultured hippocampal neurons

Neuregulin-1 (NRG-1) growth and differentiation factors and their erbB receptors are hypothesized to promote embryonic hippocampal neuron differentiation via as yet unknown mechanisms. We have found that NRG-1beta increases the outgrowth of primary neurites, neuronal area, total neurite length, and neuritic branching in E18 hippocampal neurons. NRG-1beta effects on neurite extension and arborization are similar to, but not additive with, those of brain-derived neurotrophic factor and reflect direct NRG-1 action on hippocampal neurons as these cells express the NRG-1 receptors erbB2 and erbB4, the erbB-specific inhibitor PD158780 decreases NRG-1beta induced neurite outgrowth, and NRG-1beta stimulation induces p42/44 ERK phosphorylation. Pharmacological inhibition of p42/44 ERK and protein kinase C (PKC), but not PI3K or p38 MAP kinase, inhibits NRG-1beta-induced neurite extension and elaboration. We conclude that NRG-1beta stimulates hippocampal neurite extension and arborization via a signaling pathway that involves erbB membrane tyrosine kinases (erbB2 and/or erbB4), p42/44 ERK, and PKC.     

Heparan sulfate proteoglycan and laminin mediate two different types of neurite outgrowth

Spinal cord neurons cultured in vitro have been shown to respond to changes in their environment by means of 2 different types of neurite outgrowth: (1) neurite elongation and (2) emergence and branching of newly formed neurites. Culture of spinal cord neurons with heparan sulfate proteoglycan (HSPG) medium resulted in a 3-fold increase in neurite elongation compared to the control. Extensive branching was seen when neurons were cultured in laminin-supplemented culture medium. HSPG-induced elongation and laminin-induced branching of neurites were blocked by specific anti-HSPG and antilaminin sera, respectively. Furthermore, laminin antibodies did not inhibit neurite elongation and HSPG antibodies did not block neurite branching. Conditioned medium from primary embryonic rat muscle cultures (MCM) mimicked the effects of both HSPG and laminin on neurite outgrowth. Immunoprecipitation with anti-HSPG and antilaminin antibodies demonstrated that MCM contains these 2 basal lamina components. Our observations suggest that HSPG and laminin might be highly effective molecules for promoting neurite outgrowth of rat spinal cord neurons in vitro.     

Transforming growth factor-β1 and -β2 promote neurite sprouting and elongation of cultured rat hippocampal neurons

Transforming growth factor-β (TGF-β) is known as a potent regulator of cell proliferation and differentiation. In the present study, we investigated the effects of TGF-β1 and -β2 on the survival, neurite sprouting and process elongation of primary cultured hippocampal neurons obtained from rat embryos. Addition of TGF-β1 little affected the total number of surviving neurons, but clearly increased the number of neurons bearing processes, indicating that TGF-β1 promotes neurite sprouting rather than neuronal survival. Furthermore, TGF-β1 significantly promoted the elongation of axon-like processes, but did not affect the process branching and the number of dendrite-like processes. TGF-β2 also promoted the neurite sprouting and stimulated the elongation of axons without affecting the branching. The effects of TGF-β2 were very similar to those of TGF-β1 in terms of both effective concentrations (0.1–1 ng/ml) and maximal effects. It is possible that TGF-β1 and -β2 play roles in the formation of neuritic networks in the central nervous system.     

Interleukin-2 promotes survival and neurite extension of cultured neurons from fetal rat brain

We investigated the effects of interleukin-2 (IL-2) on the survival and morphology of primary cultured neurons from fetal rat brain. Addition of recombinant human IL-2 significantly supported the survival of brain neurons in high cell density culture, but did not show any effect on the neuronal survival in low cell density culture. In addition, IL-2 significantly promoted the neurite elongation and branching of hippocampal neurons in low cell density culture. These results suggest that IL-2 supports neuronal survival indirectly and promotes neuritogenesis by directly acting on brain neurons.     

Dock4 regulates dendritic development in hippocampal neurons

Dendrite development is required for establishing proper neuronal connectivity. Rho-family small GTPases have been reported to play important roles in the regulation of dendritic growth and morphology. However, the molecular mechanisms that control the activities of Rho GTPases in developing dendrites are not well understood. In the present study we found Dock4, an activator of the small GTPase Rac, to have a role in regulating dendritic growth and branching in rat hippocampal neurons. Dock4 is highly expressed in the developing rat brain, predominantly in hippocampal neurons. In dissociated cultured hippocampal neurons, the expression of Dock4 protein is up-regulated after between 3 and 8 days in culture, when dendrites begin to grow. Knockdown of endogenous Dock4 results in reduced dendritic growth and branching. Conversely, overexpression of Dock4 with its binding partner ELMO2 enhances the numbers of dendrites and dendritic branches. These morphological effects elicited by Dock4 and ELMO2 require Rac activation and the C-terminal Crk-binding region of Dock4. Indeed, Dock4 forms a complex with ELMO2 and CrkII in hippocampal neurons. These findings demonstrate a new function of the Rac activator Dock4 in dendritic morphogenesis in hippocampal neurons.     

Early in vitro genesis and differentiation of axons and dendrites by hippocampal neurons analyzed quantitatively with neurofilament-H and microtubule-associated protein 2 antibodies

Differentiating neurons initially extend neurites that are the precursors of axons and dendrites. The temporal pattern of neurite outgrowth has been studied extensively, but mostly qualitative analyses have been used to study this phenomenon. We have examined neurite outgrowth of hippocampal neurons in primary cultures using a polyclonal antibody against microtubule-associated protein 2 (MAP2) and a novel monoclonal antibody against the phosphorylated form of high neurofilament subunit (NF-H). These antibodies serve as markers for dendrites and axons, respectively. The neurite staining patterns were quantified during the first 10 days in culture and the analysis revealed that primary processes undergo three phases of differentiation: (i) in the first 24 h, the majority of primary neurites express MAP2 only and a small percentage express both MAP2 and NF-H; (ii) between 24 and 48 h, NF-H expression increases and it is coexpressed with MAP2 in many neurites as they begin to lengthen; and (iii) between 48 h and 4 days, MAP2 and NF-H protein expression occurs in separate populations of neurites. While most of the earliest forming primary neurites appear to be dendritic (MAP2 only), the coexpression of dendritic and axonal protein markers in a group of early forming processes suggests that these neurites may not be predetermined to become a dendrite or an axon. Our data also indicate that NF-H is detectable early in primary neurite development and that, based on in vivo localization and morphology of cultured neurites, the phosphorylated form of NF-H is concentrated in axons.     

Transmembrane agrin regulates filopodia in rat hippocampal neurons in culture

Filopodia mediate axon guidance, neurite branching and synapse formation, but the membrane molecules that regulate neuronal filopodia in response to extracellular cues are largely unknown. The transmembrane isoform of the proteoglycan agrin, expressed predominantly in the CNS, may regulate neurite outgrowth, synapse formation and excitatory signaling. Here we demonstrate that agrin positively regulates neuronal filopodia. Over-expression of TM-agrin caused the formation of excess filopodia on neurites of hippocampal neurons cultured 1-6 days. Conversely, suppression of agrin expression by siRNA reduced the number of filopodia. Time lapse analysis indicated that endogenous TM-agrin regulates filopodia by increasing their stability and initiation. The N-terminal half of agrin was necessary for induction of filopodia, and over-expression of TM-agrin in a neuronal cell line increased Cdc42 activation, suggesting a role for Cdc42 downstream of agrin. By positively regulating filopodia in developing neurons, TM-agrin may influence the pattern of neurite outgrowth and synapse formation.     

Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons

A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca²⁺-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4α-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested.At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4α-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every parameter that was measured (including viability).These results suggest that these three protein kinases selectively modulate different aspects of neurite development. The universality of effects caused by calmodulin inhibition make it impossible to determine if there are specific targets of calmodulin action involved in neurite development. Finally, our data indicate that some superficially similar characteristics of neuronal differentiation, such as neurite initiation and branching, may be controlled by quite different molecular mechanisms.     

Role of vimentin in early stages of neuritogenesis in cultured hippocampal neurons

Vimentin is expressed initially by nearly all neuronal precursors in vivo, and is replaced by neurofilaments shortly after the immature neurons become post-mitotic. Moreover, both vimentin and neurofilaments can be detected transiently within the same neurite, leaving open the possibility that vimentin may play a role in the early stages of neuritogenesis. In the present study, cultured hippocampal neurons, which transiently express vimentin in culture, were treated with sense- and antisense-oriented deoxyoligonucleotides encoding regions of the vimentin sequence that overlap the translation initiation codon. Antisense oligonucleotide treatment reduced vimentin-immunoreactivity to background levels. Moreover, while 90–100% of cultured hippocampal neurons elaborated neurites within the first 24 hr following plating, only 24–30% did so in the presence of vimentin antisense oligonucleotides. Inhibition of neurite outgrowth was reversible following removal of antisense oligonucleotide. These findings substantiate earlier studies in neuroblastoma cells, indicating a possible role for vimentin in the initiation of neurite outgrowth.