福尔马林致痛对大鼠DRG神经元CCK表达的影响

胆囊收缩素(CCK)是一种脑肠肽,在中枢和外周神经系统广泛分布.CCK参与许多重要生理和病理生理过程,包括行为学、焦虑、学习、记忆过程、疼痛等,为探讨CCK是否参与初级感觉信息的调制,本实验运用免疫组织化学技术,观察了福尔马林致痛后对大鼠背根神经节(DRG)神经元CCK表达的影响.研究结果表明,大鼠足底注射福尔马林1h后,注射侧DRG小神经元和大、中神经元CCK表达阳性率分别为60.37%±6.92%和26.96%±4.01%,注射侧CCK表达阳性细胞的半定量光密度均值是0.236±0.012,与生理盐水组相比均显著增加,差异均有显著性.非注射侧DRG小神经元和大、中神经元CCK表达阳性率分别为25.22%±5.08%和11.73%±2.73%,非注射侧CCK表达阳性细胞的半定量光密度均值是0.173±0.002,与生理盐水组相比均无明显改变.福尔马林致痛后大鼠DRG神经元CCK表达阳性率和半定量光密度均值均增加,表明CCK在DRG和脊髓水平参与了痛觉信息传递的调制.     

胎鼠DRG神经元细胞培养方法的建立

目的建立胎鼠背根神经节(dorsal root ganglion,DRG)神经元原代培养的方法。方法无菌条件下取E16天的胎鼠DRG进行原代培养,观察神经元生长状态并用神经元特异性烯醇化酶(neuronal specific enolase,NSE)免疫细胞化学染色鉴定细胞。结果培养的DRG神经元可存活1个月左右,并长出突起,形成密集的网络。NSE鉴定细胞阳性表达率高,神经元达到90%左右纯度。结论本文建立了DRG神经元细胞简洁、经济、高效的培养方法并成功鉴定了神经元。为对神经元的深入研究提供了实验模型。     

糖皮质激素性骨质疏松大鼠弓状核β-内啡肽免疫反应神经元的变化

目的：观察地塞米松（DEX）致雄性大鼠骨质疏松后,其下丘脑弓状核β-内啡肽（β-endorphin,β-EP）免疫反应神经元的变化。方法：选用4月龄健康雄性SD大鼠16只,采用随机数字表法将大鼠随机分为2组,模型组给予地塞米松磷酸钠注射液以0．1mg／100g体重,肌肉注射,2次／周,连续8周。对照组肌肉注射等量生理盐水,8周后处死取材。通过检测大鼠胫骨上段骨组织形态计量学的变化判断骨代谢的情况;采用免疫组织化学方法观察弓状核β-EP免疫反应神经元的变化。结果：与对照组相比,模型组大鼠骨小梁面积百分数、骨小梁宽度和骨小梁数量明显减小（P〈0．05）,骨小梁分离度明显增大（P〈0．01）;模型组大鼠下丘脑弓状核β-EP免疫反应阳性神经元的数量明显减少（p〈0．05）。结论：弓状核β-EP免疫反应阳性神经元数量的减少可能与糖皮质激素性骨盾疏松的形成具有密切的美系。     

脑源性神经营养因子前体对海马神经元发育的影响

目的:通过体外培养海马神经元,给予抗proBDNF血清,观察其对体外海马神经元发育及存活的影响。方法:取新生0~1h的SD大鼠海马组织,体外培养神经元,给予抗pr0BDNF羊血清处理进行形态学观察,通过细胞免疫组织化学方法观察。结果:体外培养的海马神经元生长有明显的阶段性。给予抗pr0BDNF处理后,其表达量与神经元生长状态、存活细胞数成正比。培养Id组的表达水平高于3d、7d组,差值具有统计学意义。结论:给予外源性抗pr0BDNF羊血清处理后,体外培养的海马神经元生长状态良好且突起数目增多,提示内源性的proBDNF抑制神经元的发育,促进凋亡,给予外源性抗体后,减弱其生物学作用,神经元发育抑制因素减弱,存活细胞数、突起数增多。     

nNOS参与阻断5-HT_(2A)受体导致的炎症大鼠痛觉低敏

通过建立大鼠足底角叉菜胶炎症模型,在致炎1 h于炎症部位注射酮色林或其溶剂DMSO,或在18 h于炎症局部注射5-HT或其溶剂生理盐水,致炎24 h灌流取脊髓和DRG,免疫组化法显示脊髓背角和DRG中nNOS的表达.结果显示酮色林组DRG中的中、小nNOS阳性神经元百分数显著少于DMSO组,脊髓中阳性神经元数也显著少于DMSO组;而5-HT组脊髓中nNOS阳性神经元数显著高于生理盐水组,说明nNOS参与了阻断5-HT2A受体导致的炎症大鼠痛觉低敏.     

Cdk5及p35在NGF撤退诱导的已分化PC12细胞凋亡中的作用研究

目的:探讨Cdk5及p35在神经生长因子(NGF)撤退诱导的PC12细胞凋亡中的作用机制.方法:建立NGF撤退诱导的已分化PC12细胞凋亡模型,Western blotting检测Cdk5及p35在凋亡过程中表达变化情况,利用Cdk5特异性抑制剂Roscovitine预处理已分化PC12细胞,检测其对NGF撤退诱导的凋亡作用影响,向已分化PC12细胞转染真核表达质粒pCMV-p35-IRES-Cdk5,检测过表达CdkS/p35对PC12细胞凋亡的影响.结果:NGF撤退36h会引起已分化PC12细胞出现典型的DNA Ladder凋亡特征,MTT检测结果也显示,NGF撤退对PC12细胞的损伤呈时间依赖性;Roscovitine预处理已分化PC12细胞可以抑制NGF撤退诱导的细胞凋亡率,但不影响Cdk5/p35蛋白表达水平;向已分化PC12细胞中转染真核表达质粒后,能检测到Cdk5/p35蛋白的过表达,并引起PC12细胞出现凋亡样改变.结论:Cdk5及p35的活化与NGF撤退诱导的已分化PC12细胞凋亡过程密切相关,抑制Cdk5的活化有抑制细胞凋亡保护神经元的作用.     

脑内移植NGF诱导的PC-12细胞治疗大鼠帕金森病的研究

实验利用SD大鼠复制6-羟多巴胺(6-hydroxydopamine,6-OHDA)完全损伤型帕金森动物模型,将神经生长因子(nerve growth factor,NGF)处理后的肾上腺嗜铬细胞瘤细胞(plleoclxromocytoma cells,PC-12)移植人模型大鼠纹状体内,观察模型动物行为改善和移植PC-12细胞的存活情况.经行为学检测,6-OHDA动物模型复制成功率达55.1%.免疫组织化学、蛋白印迹检测结果显示模型动物黑质多巴胺能神经元数目减少,黑质酪氨酸羟化酶含量降低证明模型动物稳定、可靠.PC-12细胞经NGF(50 μg/L)连续诱导7 d,细胞逐渐平展、细胞膜变皱褶等神经元形态学特征出现后,于纹状体内行细胞移植术,经阿普吗啡(apomorphine,APO)诱导旋转行为有明显改善,蛋白印迹检测也发现移植侧具有显著的酪氨酸羟化酶免疫阳性信号.因此,NGF诱导后的PC-12细胞可以作为治疗帕金森的一种细胞供体.     

NGF诱导PC12细胞神经元样细胞分化

目的:建立PC12细胞的神经元样细胞分化模型,并探讨ERK蛋白在PC12细胞神经元样细胞分化中的可能机制.方法:以10、20、50 g/L的NGF(NGF溶于PBS,培养基中PBS的终浓度不超过2%)培养PC12细胞,应用倒置相差显微镜、显微镜测微尺及流式细胞仪鉴定PC12细胞的分化,以确定NGF使用剂量.运用免疫印迹检测不同浓度、不同作用时间时ERK蛋白在PC12细胞中的表达,并进行统计学分析.结果:随NGF剂量的升高,PC12细胞的体积、最长突起长度和突起数目均会增大或增多。统计学分析证明50 g/L的NFG作用48 h足以诱导PC12细胞的交感神经样改变。尽管ERK总蛋白水平在NFG作用前后无明显改变,但在NFG作用5 min后磷酸化的ERK蛋白水平即显著升高,达到峰值,并持续约1 h.50 g/L的NFG作用于PC12细胞48 h可使细胞发生明显的G1期阻滞.结论:PC12细胞可在NGF作用下出现交感神经样改变,并且细胞的分化程度依赖于NGF的使用剂量和作用时间.在NGF诱导的PC12细胞神经元样分化中ERK蛋白的磷酸化对NGF呈现剂量和时间依赖性,提示ERK蛋白在分化的早期发挥重要作用.     

全脑短暂性缺血后大鼠海马组织神经营养因子的时序性表达

探讨大鼠脑短暂性缺血后内源性神经营养因子NGF、BDNF和NT-3在海马组织中的变化规律,为神经损伤的修复治疗提供参考数据。本研究采用夹闭双侧颈总动脉的方法制作TGI大鼠模型,将大鼠随机分为术后3、7、14、21d以及假手术组5组,采用RT-PCR法检测大鼠海马组织中NGF、BDNF和NT-3mRNA表达水平的变化。研究结果表明3种神经营养因子在全脑短暂性缺血海马组织中出现表达下降趋势,其中以NGF mRNA的表达量下降最为显著(P<0.01)且具有快速恢复趋势,到损伤后21dNGF已上升至伤后3d水平。结果显示,这种变化规律提示短暂性脑缺血后3种神经营养因子表达水平的下降加剧了缺血再灌注对神经元的损害,其中NGF可能是脑缺血后发挥神经损伤修复的主要因子,有助于神经损伤的修复作用。     

红藻氨酸致癫痫大鼠不同时点神经生长因子变化研究

摘要: 目的 探讨红藻氨酸( Kainic Acid,KA) 致癫痫大鼠癫痫发作过程中海马组织神经生长因子( nerve growth factor,NGF) 不同时间点表达量的变化。方法 腹腔注射 KA 建立大鼠癫痫模型。采用 TaqMan 探针实时定量 PCＲ ( Ｒeal-time quantitative PCＲ) 技术,检测注射 KA 后不同时点大鼠海马组织中 NGF 表达量的变化。结果 与 NGF 表达量与生理盐水对照组( NS) 相比,6 ～ 12 h NGF 表达量明显低于 NS 组( P ＜ 0. 05) 、48 ～ 72 h NGF 表达量开始升高并高于 NS 组,在 24、72 h 时其表达量显著高于 NS 组( P ＜ 0. 05) 。结论 NGF 对致癫大鼠的海马具有修复与保护作用。     

芬必得对大鼠腰椎间盘突出症模型的疗效评价

目的 研究环氧化酶抑制剂芬必得对腰椎间盘突出症的作用。方法 通过压迫背根神经节及移植自体尾椎髓核至腰椎对大鼠进行造模。COX(cyclooxygenase)抑制剂芬必得给药剂量为0.051 g/kg体质量,连续28 d。对动物进行运动功能评分、机械刺激回缩反应测定、动物大腿周径测量及步态测量。给药28 d后取腰椎(L4～L6)及其神经用4%甲醛溶液固定后进行病理组织学检查,并取血清及神经根病变组织进行炎性因子浓度测定。结果 COX抑制剂芬必得能够改善模型动物的动物运动功能评分、50%机械性缩足反射阈值、大腿周径比(造模侧/对侧)、坐骨神经功能指数(SFI),背根神经节及椎间盘组织病理改变,降低血清和病变位置的炎症因子(PGE2、COX-2、NO、IL-1β、IL-6)的水平。结论 COX抑制剂可改善大鼠腰椎间盘突出症模型症状。     

Stimulation of specific GTP binding and hydrolysis activities in lymphocyte membrane by interleukin-2

Interleukin-2 (IL-2) is a polypeptide growth factor which stimulates the proliferation and differentiation of T lymphocytes. The receptor for IL-2 is expressed on activated T lymphocytes, cloned IL-2 dependent cells and several other cell types. Analysis of the primary structure and of immune-precipitated receptor suggests that this molecule has no intrinsic signal transduction function, unlike other growth factors. IL-2 interaction with a high affinity receptor has been shown, however, to activate the calcium/phospholipid-dependent protein kinase C (PK-C) presumably via phosphoinositide hydrolysis. Members of a family of closely related guanine nucleotide binding proteins (G proteins) regulate a diverse group of metabolic events. Two of them, Gs and Gi, stimulate and inhibit adenylate cyclase activity respectively, and other G proteins are involved in diverse signal transduction system. Another member, Go, has no known function and activation of phospholipase C has been attributed to the action of an unidentified G protein, Gp. Since it has been observed that IL-2 inhibits the catalytic activity of adenylate cyclase and that agents such as PGE2 which stimulate adenylate cyclase activity inhibit the lymphoproliferative response to IL-2, association of GTP binding proteins with IL-2 signal transduction was investigated. In this report we describe for the first time the participation of a GTP binding protein in the action of a polypeptide growth factor, interleukin-2.     

Nerve regeneration factor promotes nerve regeneration in rat

Nerve regeneration factor (NRF) extracted from an oral liquid of traditional Chinese medicine, as a nerve growth decoction has been reported by previous studies to exert effects of promoting nerve growth and preventing neuron apoptosis. For new insights into the function of NRF on primary cultured neurons, we investigated the neurite outgrowth in cultured rat dorsal root ganglion (DRG) explant treated with NRF by immunofluorescence and the gene and protein expressions of neurofilament-H(NF-H) and growth associated protein 43 (GAP43) in the cultured rat DRG neurons by real-time quantitative RT-PCR and Western blotting, respectively. In addition, we used a rat model of sciatic nerve crush to evaluate the effect of NRF on regeneration of injured sciatic nerve by a combination of walk track analysis, electrophysiological and histological assessments. The in vitro experiments indicated that NRF promoted the neurite growth of DRGs and the expression of NF-H and GAP43 at mRNA and protein levels in DRG neurons, and in vivo experiments showed that NRF improved peripheral nerve regeneration and functional recovery.     

Functional regeneration of sensory axons into the adult spinal cord

The arrest of dorsal root axonal regeneration at the transitional zone between the peripheral and central nervous system has been repeatedly described since the early twentieth century. Here we show that, with trophic support to damaged sensory axons, this regenerative barrier is surmountable. In adult rats with injured dorsal roots, treatment with nerve growth factor (NGF), neurotrophin-3 (NT3) and glial-cell-line-derived neurotrophic factor (GDNF), but not brain-derived neurotrophic factor (BDNF), resulted in selective regrowth of damaged axons across the dorsal root entry zone and into the spinal cord. Dorsal horn neurons were found to be synaptically driven by peripheral nerve stimulation in rats treated with NGF, NT3 and GDNF, demonstrating functional reconnection. In behavioural studies, rats treated with NGF and GDNF recovered sensitivity to noxious heat and pressure. The observed effects of neurotrophic factors corresponded to their known actions on distinct subpopulations of sensory neurons. Neurotrophic factor treatment may thus serve as a viable treatment in promoting recovery from root avulsion injuries. I     

Serotonin stimulates DNA synthesis in fibroblasts acting through 5-HT1B receptors coupled to a Gi-protein

Growth factors can be divided into two classes which act through distinct signal transduction pathways. One class including epidermal growth factor, platelet derived growth factor and fibroblast growth factor activates receptor tyrosine kinases, and the second class, including thrombin, bombesin, bradykinin and vasopressin activates a phosphoinositide-specific phospholipase C through GTP-binding proteins which can be inactivated by pertussis toxin. In Chinese hamster lung fibroblasts, thrombin-induced mitogenicity seems to correlate well with phospholipase C activation and both events are sensitive to pertussis toxin. Thrombin, like the other mitogens in this class, simultaneously inhibits adenylate cyclase. This involves an inhibitory G protein (Gi), a well established pertussis toxin substrate. The relative contributions of the two signalling pathways to mitogenicity has not been evaluated so far. We report here that the neurotransmitter serotonin (5-hydroxytryptamine), a contracting agent and mitogen for smooth muscle cells, activates phospholipase C, inhibits adenylate cyclase and stimulates DNA synthesis in fibroblasts. These events are sensitive to pertussis toxin. We show that the mitogenicity of 5-hydroxytryptamine can be uncoupled from phospholipase C activation that is mediated by 5-HT2 receptors, but correlates perfectly with inhibition of adenylate cyclase through 5-HT1B receptor. We propose that inhibition of adenylate cyclase or activation of an undefined effector system by Gi is important in 5-hydroxytryptamine induced DNA synthesis and contributes to the strong mitogenicity of the other members of this family of growth factors.     

蛇毒神经生长因子的分离纯化及鉴定

采用Ｓｅｐｈａｄｅｘ Ｇ５０、ＣＭ－Ｃｅｌｌｕｌｏｓｅ ３２柱层析，从中华眼镜蛇中分离出神经生长因子（Ｎｅｒｖｅ Ｇｒｏｗｔｈ Ｆａｃｔｅｒ，ＮＧＦ）。经ＳＤＳ－聚丙烯酰胺凝胶电泳及Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ法证明所得以的ＮＧＦ为单一组分，相对分子质量约为１．３×１０＾４。经凝胶等电聚焦电泳测得ＮＧＦ等电点ＰＩ约为７．０左右。经ＨＰＬＣ及电泳图像分析系统测得纯度为９８％。此ＮＧＦ等８ｄ鸡胚背     

Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1

The capacity of the adult brain and spinal cord to repair lesions by axonal regeneration or compensatory fibre growth is extremely limited. A monoclonal antibody (IN-1) raised against NI-220/250, a myelin protein that is a potent inhibitor of neurite growth, promoted axonal regeneration and compensatory plasticity following lesions of the central nervous system (CNS) in adult rats. Here we report the cloning of nogo A, the rat complementary DNA encoding NI-220/250. The nogo gene encodes at least three major protein products (Nogo-A, -B and -C). Recombinant Nogo-A is recognized by monoclonal antibody IN-1, and it inhibits neurite outgrowth from dorsal root ganglia and spreading of 3T3 fibroblasts in an IN-1-sensitive manner. Antibodies against Nogo-A stain CNS myelin and oligodendrocytes and allow dorsal root ganglion neurites to grow on CNS myelin and into optic nerve explants. These data show that Nogo-A is a potent inhibitor of neurite growth and an IN-1 antigen produced by oligodendrocytes, and may allow the generation of new reagents to enhance CNS regeneration and plasticity.     

Induction of c-fos-like protein in spinal cord neurons following sensory stimulation

It has been suggested that the proto-oncogenes c-fos and c-myc participate in the control of genetic events which lead to the establishment of prolonged functional changes in neurons. Expression of c-fos and c-myc are among the earliest genetic events induced in cultured fibroblast and phaeochromocytoma cell lines by various stimuli including growth factors, peptides and the intracellular second messengers diacylglycerol, cAMP and Ca2+. We report here that physiological stimulation of rat primary sensory neurons causes the expression of c-fos-protein-like immunoreactivity in nuclei of postsynaptic neurons of the dorsal horn of the spinal cord. Activation of small-diameter cutaneous sensory afferents by noxious heat or chemical stimuli results in the rapid appearance of c-fos-protein-like immunoreactivity in the superficial layers of the dorsal horn. However, activation of low-threshold cutaneous afferents results in fewer labelled cells with a different laminar distribution. No c-fos induction was seen in the dorsal root ganglia, gracile nucleus or ventral horn. Thus, synaptic transmission may induce rapid changes in gene expression in certain postsynaptic neurons.     

Varicella-zoster virus DNA in human sensory ganglia

Varicella-zoster virus (VZV) causes chickenpox and shingles. Clinical and epidemiological evidence indicates that following an episode of childhood chickenpox (varicella), VZV becomes latent, presumably in dorsal root ganglia, and is reactivated many years later to produce shingles (zoster) in adults. VZV has been demonstrated in ganglia by electron microscopy and by indirect immunofluorescence, and infectious viral particles have been isolated from acutely infected ganglia of patients who died of disseminated VZV infection. However, VZV has not been detected in the ganglia of humans without recent exposure to VZV. Tissue culture explant methods that have been successful in the isolation of herpes simplex virus from ganglia have so far failed in the isolation or reactivation of VZV from trigeminal and other dorsal root ganglia. We describe here the detection of VZV DNA sequences in an acutely infected human sacral ganglion and in normal trigeminal ganglia. These findings support the hypothesis that VZV is latent in normal human ganglia.     

Central nervous system and peripheral nerve growth factor provide trophic support critical to mature sensory neuronal survival

Primary sensory neurones in cranial and dorsal root ganglia (DRG) of adult animals are generally thought to be maintained through connections with their peripheral (but not central) targets by trophic factor(s) other than nerve growth factor (NGF). Damage to the peripheral process of sensory neurones results in a dramatic response or even death of the neurones, whereas axotomy (cutting) of the central process does not initiate profound reaction in these neurones. The development and maintenance of neurones are highly dependent on a supply of trophic agents produced by targets and retrogradely transported via the peripheral process to the cell body. NGF deprivation in fetal rodents produced either by exogenously administered antibodies or by those of maternal origin, results in death of DRG and of some cranial sensory neurones. However, as chronic NGF deprivation in neonatal or adult rodents produces little or no cell death, it has been assumed that some other trophic factor(s) derived from the peripheral target sustains sensory neurones in postnatal life. By inducing NGF deprivation by autoimmunizing guinea pigs with mouse NGF and/or by cutting the central root (process) of a DRG, we demonstrate here that under certain conditions DRG neurones require NGF and centrally derived trophic support. Our results indicate that sensory neurones are maintained by the trophic support provided by both peripheral and central targets. This support is mediated by NGF and other as yet unidentified trophic factors. The relative importance of the two target fields and NGF compared with other trophic factors changes during development.