NGF,BDNF和NT-3在培养鸡胚背根节神经元的表达

目的　探讨NGF ,BDNF ,NT - 3在体外培养鸡胚背根节神经元中的表达变化。方法　采用NGF ,BDNF ,NT - 3的兔抗血清分别对培养前后的鸡胚背根节神经元以免疫组化ABC法染色。观察NGF、BDNF和NT - 3在培养前、后鸡胚背根节神经元的表达情况 ,计数并比较培养前、后三种因子免疫阳性神经元百分数。结果　未培养的神经元 ,NGF ,BDNF ,NT - 3的阳性神经元百分数分别是 :10 %± 3%,2 7%± 5 %,2 9%± 7%。培养 48小时后 ,NGF ,BDNF ,NT - 3的阳性神经元百分数分别是 :77%± 6 %,6 4%± 7%,2 4%± 7%。结论　培养后NGF ,BDNF的表达较未培养者增加 (P <0 0 1) ,而NT - 3者则有减少 (P <0 0 5 )。提示在体外培养的鸡胚背根节神经元NT - 3的表达有不同于NGF和BDNF的调节方式。     

NGF 对大鼠胚胎中脑神经细胞体外增殖和分化的影响

目的:探讨神经生长因子(nerve growth factor,NGF)对大鼠胚胎中脑神经细胞体外增殖和分化的影响。方法:在体外分离培养大鼠胚胎中脑神经细胞的培养液中加入不同浓度(10、50、100、200ng/ml)的NGF,培养不同时间,以不加神经营养因子的细胞为对照组,通过MTT法检测细胞活性,神经元特异性烯醇化酶免疫细胞荧光技术鉴定神经细胞,光镜下形态学观察各组大鼠中脑神经细胞体外增殖和分化情况。结果:胚胎中脑神经细胞胞体增大、突起延长且有丰富的神经纤维连结成网络状,细胞集落数增加,显示出剂量-效应关系。结论:一定剂量的NGF能促进大鼠中脑神经细胞分化和增殖,增强其活性。     

人神经生长因子的原核高效表达

神经生长因子(nerve growth factor,NGF)是神经系统最重要的生物活性分子之一,也是最早发现和最典型的神经营养因子。它影响外周和中枢神经系统某些神经元的存活与分化;NGF在神经损伤时可保护其效应神经元,促进神经纤维再生,增加脑移植中某些神经元的存活;它对外伤、中毒、老化等因素引起的脑疾患有治疗作用,并具有神经修复功能,尤其是对早老性痴呆症、帕金森氏病的治疗作用比较乐观。虽然NGF在神经损伤和神经退行性病变的诊断和治疗中有巨大的临床应用价值,但天然NGF受到雄性小鼠颌下腺这     

神经生长因子及其受体相关信号传导通路的研究进展

神经生长因子是神经营养因子家族成员之一,对不同时期神经元的存活、分化、生长及损伤后的修复和再生都有着十分重要的作用。不仅在神经系统中,随着人类的其他正常和肿瘤组织中同样也检测得到了NGF,神经生长因子在各方面的应用也得到了重视并均已得到了证实。NGF功能的发挥离不开与其受体的结合,根据NGF表面糖蛋白与凝集素结合能力的不同,其受体可被分为高亲和力受体酪氨酸激酶A和低亲和力受体p75。Trk A与NGF结合后所介导的信号通路主要有:1MAPK通路;2PLC-γ通路;3PI3K/PKB通路。而p75与NGF结合介导的信号传导通路主要包括:1NF-κB通路;2JNK-p53-Bax凋亡通路;3神经酰胺通路。Trk A一般介导的是正性信号,如促进神经细胞生长、维持神经细胞的存活等;而p75既可促进神经细胞存活,也可诱导神经细胞凋亡,但以后者为主。当Trk A与p75同时表达时,Trk A可抑制p75诱导细胞凋亡,使受损神经细胞大量增殖,所以其生物学总效应是促进神经细胞的生长和存活。     

LRRN3膜蛋白在人胚脊神经节的分布和表达

目的:探讨神经系统富亮氨酸重复超家族成员LRRN3膜蛋白在人胚脊神经节的表达和分布。方法:从人胚脊神经节分离mRNA和蛋白质,用RT-PCR、Western印记杂交法和免疫组织化学方法检测LRRN3膜蛋白表达。结果:LRRN3膜蛋白C端序列RT-PCR扩增产物cDNA在各胎龄脊神经节均有表达,长度约500bp;Western印记杂交结果显示LRRN3膜蛋白存在,分子质量约78 kD;免疫组织化学和免疫荧光组织化学结果表明LRRN3阳性表达细胞均为脊神经节感觉神经细胞,部分神经细胞弱阳性表达,部分未表达。结论:LRRN3膜蛋白在脊神经节神经元的表达,推断与脊神经节感觉神经元的发育、形态构建及损伤后修复有密切的关系。     

LRRN3膜蛋白在人胚脊神经节的分布和表达

目的：探讨神经系统富亮氨酸重复超家族成员LRRN3膜蛋白在人胚脊神经节的表达和分布。方法：从人胚脊神经节分离mRNA和蛋白质,用RT-PCR、Western印记杂交法和免疫组织化学方法检测LRRN3膜蛋白表达。结果：LRRN3膜蛋白C端序列RT—PCR扩增产物cDNA在各胎龄脊神经节均有表达,长度约500bp;Western印记杂交结果显示LRRN3膜蛋白存在,分子质量约78kD;免疫组织化学和免疫荧光组织化学结果表明LRRN3阳性表达细胞均为脊神经节感觉神经细胞,部分神经细胞弱阳性表达,部分未表达。结论：LRRN3膜蛋白在脊神经节神经元的表达,推断与脊神经节感觉神经元的发育、形态构建及损伤后修复有密切的关系。     

银杏内酯对胚基底前脑NOS、AChE阳性神经元发育的影响

目的 探讨银杏内酯对胚基底前脑NOS和AChE阳性神经元发育的影响。方法 实验分成银杏组,NGF组,BDNF组和单纯对照组。取孕17dSD大鼠胚基底前脑原基制成细胞悬液接种于96孔培养板和2块24孔培养板中,分别加入含银杏内酯,NGF,BDNF及不含上述成份的DMEM培养液,于体外培养18d后,96孔培养板行MTT比色分析测定光吸收值（OD值）,以检测培养的神经元活力,2块24孔培养板分别行NADPH-d和AChE组化染色,显微镜下计数各组每了忆中的NOS和AChE阳性神经元数,并用CMM-301图像分析系统对两种神经元的细胞面积和细胞周长进行处理。数据用方差分析和SNK检验进行统计学处理。结果 银杏组MTT比色分析的OD值和NOS,AChE阳性神经元数,细胞面积,细胞周长等指标均明显地好于单纯对照组,达到或仅稍差于NGF组或BDNF组的指标。结论 银杏内酯在促进胚基底前脑NOS和AChE阳性神经元发育方面具有类似NGF和BDNF的作用。     

新生鼠大脑皮质和中脑多巴胺神经元原代培养中NGF诱导c-jun mRNA表达的研究

用神经生长因子（nervegrowthfactor,NGF）分别处理原代培养的新生大鼠大脑皮质和中脑腹侧部神经元,应用免疫组织化学和原位杂交双重标记方法,观察不同时间NGF处理的神经元表达原癌基因c－junmRNA的情况。结果发现,神经特异性烯醇化酶（neuronspecificenolase,NSE）阳性的大脑皮层神经细胞在NGF处理15分钟即可表达c－junmRNA,2小时达高峰,4小时后开始下降,到8小时后基本消失．未经NGF处理的大鼠大脑皮层神经细胞不表达c－junmRNA;酪氨酸羟化酶（tyrosinehydroxylase,TH）阳性的中脑多巴胺能（Dopaminergic,DA）神经元经NGF处理也不表达c－jun基因。提示NGF与其受体结合可以激活神经细胞快速,短暂的一过性表达c-jun基因,作为第三信使,调节从细胞质膜到核的信号传递,同时也间接证明了新生大鼠大脑皮层神经细胞膜上存在神经生长因子受体（nervegrowthfactorreceptor,NGFR）,而中脑DA神经细胞对NGF无应答反应。     

低氧预处理对大鼠海马神经元缺氧耐受性和Jun基因表达的影响

目的 :观察低氧预处理对缺氧 复氧后大鼠海马神经元Jun表达的影响。方法 :取培养 12d的两组 (对照组和低氧预处理组 )神经元 ,同时置于缺氧环境 (0 .90L LN2 0 .10L LCO2 )中培养 4h后取出 ,置含 0 .10L LCO2 和空气的培养箱内复氧培养 2 4h和 72h ,于不同时间取出 ,观察神经元存活数 ,并用抗Jun抗血清进行免疫组织化学染色 ,观察Jun表达阳性和阴性神经元数目 ,计算Jun表达神经元所占百分率。结果 :经低氧预处理的海马神经元缺氧 复氧后Jun表达阳性神经元百分率较对照组明显减少 ,神经元存活数明显高于对照组。结论 :低氧预处理可使海马培养神经元对缺氧产生耐受 ,减少缺氧 复氧后神经元Jun的表达。提高神经元存活数     

rhCNTF对鸡胚感觉与运动神经元神经营养作用的比较

在无血清培养条件下,观察了重组人睫状营养因子（ｒｈＣＮＴＦ）对鸡胚背根节感觉神经元及腹角运动神经元的营养作用。结果表明ｒｈＣＮＴＦ对这两类神经元均有明显的促存活作用,并呈一定的剂量／效应关系。ｒｈＣＮＴＦ浓度在０．５ｎｇ／ｍｌ以下时无作用,１．０－１．５ｎｇ／ｍｌ时已有促神经元存活作用,４ｎｇ／ｍｌ时作用最明显,再增加到１００ｎｇ／ｍｌ神经元存活数无进一步增加。比较培养７天时两类神经元存活数发现感觉神经元对ＣＮＴＦ缺乏的敏感性高于运动神经元,提示ＣＮＴＦ对运动神经元的促存活作用只是它多种类型神经元营养作用中较弱的一环     

Stage-dependent stimulation of neurite outgrowth exerted by nerve growth factor and chick heart in cultured embryonic ganglia

The ability of embryonic chick heart to elicit neuritic outgrowth in different ganglia was tested to examine (1) whether stimulative activity is possessed by the heart only at specific stages and (2) whether the ability of the ganglionic neurons to respond is limited to certain periods of development. As an assay, ganglia were explanted into thin collagen gels with ventricular tissue placed at a distance of about 1 mm. Neuritic outgrowth was measured after 2 days. Control ganglia and ganglia cultured with added nerve growth factor (NGF) were also scored. Four types of tested ganglia, including the ciliary ganglion, showed a peak in neuritic outgrowth when cultured with heart of embryonic Day 18, at about which age the heart becomes sympathetically innervated in ovo. No age-related size differences that could account for this temporal pattern were found among the heart explants when measuring their protein content. A peak in neuronal susceptibility to heart tissue was evident in the 6-day ciliary ganglion and in the 8-day paravertebral, Remak, and spinal ganglia, roughly coinciding with the onset of fibre outgrowth in ovo. Neurite extension is concluded to have been triggered by a factor spread from the heart explants and being distinct from the mouse type of NGF since anti-NGF did not at any stage block the events and since added NGF at all stages failed to evoke neurite formation in the ciliary ganglia. A testable hypothesis is that this factor regulates the growth of sympathetic and possibly parasympathetic and sensory fibres in the developing chick heart.     

Fate of intraocular chromaffin cell suspensions: role of initial nerve growth factor support

Summary Adrenal medullary tissue from adult rats was dissociated into cell suspensions and injected into the anterior chamber of the eye, where the cells were made to attach to the previously sympathectomized irides with the use of fibronectin. Short- and long-term survival of the chromaffin cells was examined in whole mounts of irides using Falck-Hillarp fluorescence histochemistry or indirect immunohistochemistry with antibodies against adrenaline and dopamine--hydroxylase (DBH). After 6 days in oculo all cells were immunoreactive for adrenaline; almost none displayed processes even if -nerve growth factor (NGF) was given at grafting. One month after weekly intraocular injections of NGF, many cells were surrounded by nerve fiber net-works and all cells were DBH-immunoreactive. Eight months postgrafting and 7 months after the last injection of NGF almost the entire iris was reinnervated and resembled a normal, sympathetically innervated iris. Both at 1 and 8 months, chromaffin cells, ganglion cells and transitional cell forms (chromaffin cells transforming towards ganglion-like cells) were found in irides from the NGF-treated eyes. The number of ganglion cells was remarkably increased with time by NGF, while the number of chromaffin cells decreased compared to controls. A single treatment with NGF at grafting had no marked effects as examined up to 3 months; at this time there was a certain outgrowth of nerve terminals, which, however, was not as pronounced as 1 month after repeated NGF injections. In conclusion, it is shown that some cells in a chromaffin cell suspension attach to the iris, transform to ganglion cells after an induction with exogenous NGF, and reinnervate the sympathically denervated iris. Such cells remain ganglion-like in character and continue to form processes even after cessation of exogenous NGF treatment.     

Retrograde Transport of Endogenous Nerve Growth Factor in Superior Cervical Ganglion of Adult Rats

The concentration of naturally synthesized nerve growth factor (NGF) was measured in various tissues of adult rats, using a highly sensitive two-site enzyme immunoassay. The highest concentration was found in the superior cervical sympathetic ganglion (SCG). Transection of the postganglionic external carotid nerve (ECN) reduced the ganglionic level of NGF more than did section of the internal carotid nerve (ICN). When both the preganglionic nerve and the ECN were cut, the ganglionic NGF level decreased even more. On the other hand, when the preganglionic nerve and the ICN were both sectioned, leaving the ECN intact, endogenous NGF content in the SCG was significantly enhanced 3-9 h after operation. Bilateral extirpation of submaxillary gland produced a rapid decrease in ganglionic NGF 3-6 h after operation, and even unilateral removal of one salivary gland caused a decrease in both ganglia, which was however much greater in the ipsi- than in the contralateral ganglion. Removal of the eyeballs caused a much smaller reduction in ganglionic NGF than did removal of the glands. These results suggest that the endogenous NGF that accumulates in the SCG is mostly synthesized in the submaxillary gland rather than in the iris, and that it is transported to the SCG, mostly via the ipsilateral ECN.     

Nerve growth factor levels in mouse serum: Variations due to stress

The presence of nerve growth factor (NGF) in the serum of adult male mice was assayed using the chick embryo dorsal root ganglion (DRG) bioassay technique in a serum free N1 supplemented medium. Wide variations in the serum-induced nerve fiber outgrowth response were observed when serum was obtained from animals maintained four per cage. Of 64 mice tested, sera of 7 animals induced a profound nerve fiber outgrowth response while the sera of 57 mice failed to show a similar response. In animals kept in isolation for 7 days prior to the start of the experiment, aggression provoked a marked increase in serum NGF levels. In contrast to the sera of aggression-unprovoked mice, the sera of all aggression-provoked mice stimulated a dense nerve fiber outgrowth. The sera of both groups of mice stimulated an intense proliferation and migration of nonneuronal cells. The neurite outgrowth responses elicited by sera from aggression-provoked and unprovoked mice were completely inhibited by the rabbit anti-NGF antiserum. In conclusion, both crowded housing and aggression in mice may provoke an elevation in the serum NGF levels that can be confirmed by the ganglion bioassay technique.     

Nerve growth factor (NGF) regulates adult rat cultured dorsal root ganglion neuron responses to the excitotoxin capsaicin

An overlap between subpopulations of nerve growth factor (NGF)-responsive and capsaicin-sensitive dorsal root ganglion (DRG) sensory neurons has been suggested from a number of in vivo studies. To examine this apparent link in more detail, we compared the effects of capsaicin on adult rat DRG neurons cultured in the presence or absence of NGF. Capsaicin sensitivity was assessed histochemically by a cobalt staining method, by measuring capsaicin-induced 45Ca2+ uptake, and by electrophysiological recording of capsaicin-evoked membrane currents. When cultured with NGF, approximately 50% of these adult DRG neurons were capsaicin-sensitive, whereas adult sympathetic neurons or ganglionic nonneuronal cells were insensitive. DRG cultures grown in the absence of NGF, however, were essentially unresponsive to capsaicin. Capsaicin sensitivity could be regained fully within 4-6 days of replacement of NGF. These results indicate that, at least in vitro, NGF can modify the capsaicin sensitivity of adult DRG neurons.     

Growth and differentiation of PC6 cells: the effects of pulsed electromagnetic fields (PEMF)

Previous studies in our laboratory showed that neurite outgrowth in vitro and nerve regeneration in vivo were stimulated by 2 Hz, 0.3 mT (3 G) pulsed electromagnetic fields (PEMF). To learn more about the effects of PEMF on nerve cells, we exposed PC6 cells, a standard neuronal-like cell model, to the same pulsed electromagnetic fields for 2 h/day for 2 days and asked whether two different cell processes, proliferation and differentiation, were affected. The cells were also treated with a differentiating agent, nerve growth factor (NGF), to further define any interactive effects. We found that proliferation was unaffected by either PEMF or NGF alone or in combination. Differentiation, expressed as neurite outgrowth, was strongly upregulated with NGF, but this NGF response was significantly depressed in cells treated with PEMF.     

Neutrophic influence of denervated sciatic nerve of on adult dorsal root ganglion neurons

Isolated adult frog dorsal root ganglion neurons survive in vitro in a defined medium for more than 4 weeks and extend processes. When co-cultured with a 1-mm piece of peripheral nerve the average tottal process lenght per neuron was 10 times longer than that of control neurons by 8 days, and the processes had a significantly different morphology from that of control neurons. This influence on process length increased with increasing time of nerve denervation length increased with increasing time of nerve denervation prior to co-culturing. These results suggest the release of a neurotrophic factor/s from the cells of the peripheral nerve. The neurotropic influence was completely blocked by antibodies against mouse nerve growth factor (NGF). Although NGF increased the average process length by twofold over control neurons, its influence never reached that of the nerve-released factor, and the NGF-induced processes had a distinctly different morphology. The frog nerve-released factor promoted process outgrowth from E11 chick sympathetic ganglia, although the process number, length, and their fasciculation differed greatly from those induced by NGF. These results suggest that the nerve-released factor/s are immunologically and functionally related to NGF but have not estabished whether a single factor or an aggregate of several secreted molecules are responsible. This article presents a new preparation in which the varied influences of different neurotrophic factors can be studied in great detail on large populations of isolated adult vertebrte neurons and sets the stage for the characterization and isolation of the frog peripheral nerve neurotrophic factor, as well as examining the influence of this facor on neuronal morphology and its ability to direct process outgrowth. 1994 John Wiley & Sons, Inc.     

Reduction in nerve growth factor availability leads to a conditioning lesion‐like effect in sympathetic neurons

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target‐derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF‐treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Effects of Nerve Growth Factor and Phorbol Derivative on Reactivation of Herpes Simplex Virus Type 1 in Cultured Cells of Latently Infected Adult Mouse Trigeminal Ganglia

Reactivation of herpes simplex virus type 1 (HSV-1) occurred rapidly in cells of latently infected adult mouse trigeminal ganglia which were cultured in serum-free medium in the presence of sufficient nerve growth factor (NGF). However, HSV-1 reactivation was delayed significantly in ganglionic cultures in the absence of exogenous NGF or in cultures treated with 2-aminopurine in the presence of NGF. The delayed viral reactivation in ganglionic cultures without NGF was accelerated by treatment with phorbol myristate acetate or dibutyryl cyclic AMP. Culture conditions which affected HSV-1 reactivation did not affect replication of HSV-1 in normal ganglionic cultures.