NGF,BDNF,NT-3和GDNF在背根节不同神经元的配布的免疫组织化学证据

为了探讨神经生长因子、脑源性神经生长因子 ,神经营养素 -3,胶原细胞源性神经生长因子在成年猫背根节神经元不同大小细胞的分布。取 L6背根节制成 2 0μm厚冰冻切片 ,用上述四种因子抗血清进行免疫组化反应。计数每一种因子阳性大、中、小型 (小于 42μm者为小型神经元 ;42～ 5 7μm者为中型神经元 ;大于 5 7μm者为大型神经元 )神经元的百分数。结果证明 ,此四种因子在背根节大、中、小神经元的阳性百分数分别为 :1.2± 0 .7,2 .1± 0 .9,3.3± 1.1;1.7± 0 .4,11.2± 1.2 ,30 .0± 1.5 ;2 6 .7± 2 .2 ,6 .2± 1.2 ,13.2± 2 .9;2 7.4± 3.3,8.1± 1.7,2 0 .1± 2 .4。表明 :在成年猫 L6背根节仅存在少数神经生长因子阳性细胞 ,而脑源性神经生长因子则主要配布于中、小神经元 ,神经营养素 -3和胶质细胞源性生长因子的免疫阳性细胞主要是大神经元 ,但也包括一些中小神经元。提示此四种因子在成年猫背根节的生理功能可能与神经元的不同大小有关。     

神经营养因子(NGF、NT-3、BDNF及CNTF)和受体trkA、trkB、trkC在正常大鼠脊髓和胳鼠脊髓移植体的免疫组织化学研究

本研究采用免疫组织化学方法观察了NGF家族（NGF、NT－3、BDNF）和非NGF家族的CNTF以及NGF家族因子受体trkA、trkB、trkC在正常大鼠脊髓腰段的分布和胎鼠脊髓移植体在移植后4周的表达。在正常大鼠脊髓腰段,各神经营养因子及受体反应物主要存在于脊髓灰质,特别是前角的运动神经元。但在脊髓后角的分布稍有不同,BDNF阳性细胞的胞浆染色较深,胞核不染色;而NT－3的胞核染色较胞浆为深,核仁不染色。在胎鼠脊髓移植体,NGF、BDNF、CNTF和NT－3及受体trkA、trkB、trkC均有不同程度的染色。本实验结果揭示了在正常大鼠脊髓神经元内各神经营养因子及受体的表达,提示神经营养因子除具有靶源性来源以外,还有神经元自分泌的产物。而在胎鼠移植体内和其周围组织神经营养因子及其受体的表达,可能是在移植体内的移植细胞自分泌和成鼠脊髓损伤的刺激所引起的,这在移植体的存活和发育中有重要作用。     

Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.     

Neurotrophin and Neurotrophin Receptors in Vascular Smooth Muscle Cells: Regulation of Expression in Response to Injury

The neurotrophins, a family of related polypeptide growth factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3 and NT-4/5 promote the survival and differentiation of distinctive sets of embryonic neurons. Here we define a new functional role for neurotrophins, as autocrine or local paracrine mediators of vascular smooth muscle cell migration. We have identified neurotrophins, and their cognate receptors, the trk tyrosine kinases, in human and rat vascular smooth muscle cells in vivo. In vitro, cultured human smooth muscle cells express BDNF; NT-3; and trk A, B, and C Similarly, rat smooth muscle cells expressed all three trk receptors as well as all four neurotrophins. Moreover, NGF induces cultured human smooth muscle cell migration at subnanomolar concentrations. In the rat aortic balloon deendothelialization model of vascular injury, the expression of NGF, BDNF, and their receptors trk A and trk B increased dramatically in the area of injury within 3 days and persisted during the formation of the neointima. In human coronary atherosclerotic lesions, BDNF, NT-3, and NT-4/5, and the trk B and trk C receptors could be demonstrated in smooth muscle cells. These findings suggest that neurotrophins play an important role in regulating the response of vascular smooth muscle cells to injury.     

Neurotrophin system expression in human pulmonary carcinoid tumors

Neurotrophin (NT) and NT receptor expression was assessed in 12 typical (TC) and 8 atypical (AC) human pulmonary carcinoids by Western blot and immunohistochemistry. TC and AC carcinoid express to different extent NT and NT receptor proteins. Nerve growth factor (NGF) was expressed by 83% of the TC but not by the AC carcinoids. Brain derived neurotrophic factor (BDNF) was expressed by 33 and 100% of TC and AC carcinoids, respectively. NT-3 was expressed by 58% of the TC and 38% of AC carcinoids. TC carcinoids express high affinity NT receptors while 50% of the AC carcinoids express the TrkB receptor. Our results demonstrate that NGF/TrkA and BDNF/TrkB signaling need to be considered as regulatory pathways that may address survival, differentiation and/or aggressiveness of human pulmonary carcinoids. Contrarily to the BDNF/TrkB, expression of the NGF/TrkA signaling may overcome aggressiveness of carcinoid cells. NTs may be useful as markers in the clinic.     

Neurotrophin system expression in human pulmonary carcinoid tumors

Neurotrophin (NT) and NT receptor expression was assessed in 12 typical (TC) and 8 atypical (AC) human pulmonary carcinoids by Western blot and immunohistochemistry. TC and AC carcinoid express to different extent NT and NT receptor proteins. Nerve growth factor (NGF) was expressed by 83% of the TC but not by the AC carcinoids. Brain derived neurotrophic factor (BDNF) was expressed by 33 and 100% of TC and AC carcinoids, respectively. NT-3 was expressed by 58% of the TC and 38% of AC carcinoids. TC carcinoids express high affinity NT receptors while 50% of the AC carcinoids express the TrkB receptor. Our results demonstrate that NGF/TrkA and BDNF/TrkB signaling need to be considered as regulatory pathways that may address survival, differentiation and/or aggressiveness of human pulmonary carcinoids. Contrarily to the BDNF/TrkB, expression of the NGF/TrkA signaling may overcome aggressiveness of carcinoid cells. NTs may be useful as markers in the clinic.     

BDNF,NGF和NT-3的mRNA和蛋白在猫左侧腰第六背根节共表达的不同方式(英文)

本实验研究了脑源性神经营养因子(brain derived neurotrophic factor,BDNF),神经生长因子(nerve growth factor,NGF)和神经营养因子-3(neurotrophin-3,NT-3)的mRNA和蛋白在猫左侧腰第六背根节(dorsal root ganglion,DRG)共表达的不同方式,并探讨了共表达的机制,为阐明神经营养因子的表达与脊髓可塑性的关系提供依据。本实验所使用的为未接受任何处理的健康猫。猫的左侧腰第六DRG被取出,行免疫组织化学染色与原位杂交方法结合的双重标记,确定是否有BDNF,NGF和NT-3的蛋白与mRNA共表达。实验结果显示BDNF,NGF和NT-3的蛋白与mRNA在猫DRG均有表达,但这三种神经营养因子mRNA和蛋白共表达的方式是不同的。免疫组化结果显示:BDNF阳性产物主要分布于细胞质和细胞核,细胞核的染色颜色较细胞质浅;NGF阳性产物主要分布于细胞核;NT-3阳性产物主要分布于细胞质。原位杂交结果显示:BDNF和NGF阳性信号主要分布于细胞质;NT-3阳性信号在细胞质和细胞核都有分布。由此可见,BDNF,NGF和NT-3的mRNA和蛋白在猫左侧腰第六DRG有不同的共表达方式,提示它们可能存在与脊髓可塑性有关的自分泌和/或旁分泌机制。     

Investigating the synergistic effect of combined neurotrophic factor concentration gradients to guide axonal growth

Neurotrophic factors direct axonal growth toward the target tissue by a concentration gradient, which is mediated through different tyrosine kinase cell surface receptors. In this study, well-defined concentration gradients of neurotrophic factors (NFs) allowed us to study the synergistic effect of different NFs (e.g. nerve growth factor [NGF], neurotrophin-3 [NT-3] and brain-derived neurotrophic factor [BDNF]) for axonal guidance of embryonic lumbar dorsal root ganglion cells (DRGs). Effective guidance of DRG axons was achieved with a minimum NGF concentration gradient of 133 ng/ml/mm alone, or combined NGF and NT-3 concentration gradients of 80 ng/ml/mm each. Interestingly, the combined concentration gradients of NGF and BDNF did not show any significant synergism at the concentration gradients studied. The synergism observed between NGF and NT-3 indicates that axons may be guided over a 12.5 mm distance, which is significantly greater than that of 7.5 mm calculated by us for NGF alone or that of 2 mm observed by others.     

Pacinian corpuscle development involves multiple Trk signaling pathways.

The development of crural Pacinian corpuscles was explored in neonatal mutant mice lacking nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) or neurotrophin-4 (NT4), or their cognate Trk receptors. Deficits of the corpuscles and their afferents were greatest in NT3, less in BDNF, and least in NT4 null mice. Deletion of NGF or p75(NTR) genes had little or no impact. No Pacinian corpuscles were present in NT3;BDNF and NT3;NT4 double or NT3;BDNF;NT4 triple null mice. Deficits were larger in NT3 than TrkC mutants and were comparable to deficits observed in TrkB or TrkA mutants. Afferents of all corpuscles coexpressed TrkA and TrkB receptors, and some afferents coexpressed all three Trk receptors. Our results suggest that multiple neurotrophins, in particular NT3, regulate the density of crural Pacinian corpuscles, most likely by regulating the survival of sensory neurons. In addition, NT3/TrkB and/or NT3/TrkA signaling plays a greater role than NT3/TrkC signaling in afferents to developing Pacinian corpuscles.     

Neurotrophin system activation in pleural effusions

Neurotrophins (NTs) expression was assessed in malignant and non-malignant pleural effusions (inflammatory exudates and transudates). Enzyme-linked immunosorbent assay, in malignant exudates from small and non-small cell lung cancer (SCLC and NSCLC), detected nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their levels are higher as compared with inflammatory and transudative effusions. By immunoblots, in cultured cancer cells coming from malignant pleural effusions, NTs and low- and high-affinity NT receptors were detected in a percentage of SCLC and NSCLC. Proliferation assay demonstrated that BDNF significantly increased cancer cell proliferation in vitro, on the contrary, NT-3 reduced cancer cell growth rate and NGF did not modify cell growth. Moreover, NGF protects cells from death during starvation. These effects are reverted by the addition of NT receptor antagonists. Cultured cancer cells injected into the lung of immunodeficient mice generate lung tumors expressing NTs and NT receptors. These findings suggest that NTs may be able to modulate cancer cell behavior and their growth.     

Expression of insulin-like growth factor-1 receptor (IGF-1R) and p27Kip1 in melanocytic tumors: a potential regulatory role of IGF-1 pathway in distribution of p27Kip1 between different cyclins

Alveolar macrophages play a crucial role in regulating lung immune responses and in maintaining the integrity of the respiratory tract. Neurotrophins (NTs), besides to their neurotrophic activities, exhibit physiological effects in the immune system. In this study, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), NT-3 and low- (p75) and high affinity (Trks) NT receptors were investigated by immunocytochemistry in cytospin centrifuged preparations of human alveolar macrophages. Approximately 2.5% alveolar macrophages were immunoreactive for NGF, whereas no macrophages displaying immunoreactivity for BDNF or NT-3 were observed. A 3.5% macrophages displayed immunoreactivity for TrkA-receptor protein, 10% for TrkB-receptor protein (full length isoform), and 2% for TrkC-receptor protein. No low-affinity p75 NT and TrkB[-] truncated isoform receptor immunoreactive macrophages were found. These findings support the hypothesis that NTs and the corresponding receptors may play a role in regulating immunological and functional activity of alveolar macrophages via paracrine/autocrine mechanisms.     

脊髓半横断损伤后腹角神经元神经生长因子、脑源性神经营养因子、神经营养因子-3和神经营养因子-4表达的变化

目的:探讨大鼠脊髓半横断损伤(htSCI)后脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养因子(NT-3、NT-4)在脊髓腹角神经元表达的早期变化。方法:免疫组织化学ABC法分别染4种神经因子并作阳性细胞计数。结果:NGF主要分布于脊髓腹角神经元的胞核,BDNF、NT-4与NT-3主要分布于胞浆。htSCI前后它们在细胞内的分布范围没有变化。BDNF、NGF与NT-3的3 d在损伤尾侧段脊髓双侧腹角阳性神经元数与对照组相比显著减少。BDNF与NGF的14 d的双侧腹角阳性神经元数量均较正常组明显增多,NT-3与NT-4的14 d~21 d的双侧腹角阳性神经元数量均较正常组明显增多,BDNF7~21 d以及NGF14 d的健侧的阳性神经元数量均分别多于相应的伤侧。结论:内源性BDNF、NGF、NT-3、NT-4增加对脊髓损伤修复具有重要作用,BDNF和NGF在健侧表达的增加说明健侧代偿功能的活跃。     

脂肪源性干细胞修复周围神经损伤潜能的研究

目的:体外原代培养脂肪源性干细胞(adipose-derived stem cell,ADSC),并在细胞水平检测各种神经营养因子(neurotrophic factor,NF)的表达情况,探讨ADSC是否可以作为神经损伤修复的种子细胞。方法:体外原代培养和鉴定ADSC,应用RT-PCR方法检测ADSC细胞神经营养因子,包括神经生长因子(nerve growth factor,NGF)、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、神经营养素-3(neurotrophin 3,NT-3);免疫组织化学法检测粘附因子(nerve cell adhesion molecule,NCAM)表达情况。结果:ADSC可以向脂肪和骨两方面分化;RT-PCR结果显示ADSC细胞NGF、BDNF、NT-3 mRNA水平明显升高;同时免疫荧光法检测到了高水平的NCAM。结论:ADSC具有干细胞分化潜能,并且能够表达NF和NCAM。     

Neurotrophins and neurotrophin receptors in human lung cancer.

The expression of neurotrophins (NTs) and related high- and low-affinity receptors was studied in surgical samples of histologically diagnosed human tumors of the lower respiratory tract. The experiment was conducted with 30 non-small cell lung cancer specimens and in eight small cell lung cancer specimens by Western blot analysis and immunohistochemistry to assess expression and distribution of NT and NT receptor proteins in tissues examined. Immunoblots of homogenates from human tumors displayed binding of anti-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and NT-3 antibodies as well as of anti-tyrosine-specific protein kinase (Trk) A, TrkB, and TrkC receptor antibodies, with similar migration characteristics than those displayed by human beta-NGF and proteins from rat brain. A specific immunoreactivity for NTs and NT receptors was demonstrated in vessel walls, stromal fibroblasts, immune cells, and sometimes within neoplastic cell bodies. Approximately 33% of bronchioloalveolar carcinomas exhibited a strong membrane NGF and TrkA immunoreactivity, whereas 46% adenocarcinomas expressed an intense TrkA immunoreactivity but a weak immunostaining for NGF within tumor cells. Moreover, squamous cell carcinomas developed an intense TrkA immunoreactivity only within stroma surrounding neoplastic cells. A faint BDNF and TrkB immunoreactivity was documented in adenocarcinomas, squamous cell carcinomas, and small cell lung cancers. NT-3 and its corresponding TrkC receptor were found in a small number of squamous cell carcinomas within large-size tumor cells. No expression of low-affinity p75 receptor protein was found in tumor cells. The detection of NTs and NT receptor proteins in tumors of the lower respiratory tract suggests that NTs may be involved in controlling growth and differentiation of human lung cancer and/or influencing tumor behavior.     

Electro-acupuncture induced NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion in cats subjected to removal of adjacent ganglia

This study evaluated the effect of electro-acupuncture (EA) on the NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion (DRG) in cats subjected to bilateral removal of L1-L5 and L7-S2 DRG, using immunostaining, in situ hybridization and RT-PCR. The positive products of NGF, NT-3 protein and mRNA in the small and large neurons of spared L6 DRG in EA side increased greatly more than that of control side, while the increased BDNF was only noted in small and medium-sized neurons. RT-PCR demonstrated that the mRNA level for three factors was not influenced by EA in intact DRG, when a significant increase was seen in the spared L6 DRG of EA side. As it has been well known that DRG neurons project to the spinal cord wherein morphological plasticity has been present after DRG removal, the present results might have some bearing to the observed phenomenon.     

Met receptor signaling is required for sensory nerve development and HGF promotes axonal growth and survival of sensory neurons

The development of the nervous system is a dynamic process during which factors act in an instructive fashion to direct the differentiation and survival of neurons, and to induce axonal outgrowth, guidance to, and terminal branching within the target tissue. Here we report that mice expressing signaling mutants of the hepatocyte growth factor (HGF) receptor, the Met tyrosine kinase, show a striking reduction of sensory nerves innervating the skin of the limbs and thorax, implicating the HGF/Met system in sensory neuron development. Using in vitro assays, we find that HGF cooperates with nerve growth factor (NGF) to enhance axonal outgrowth from cultured dorsal root ganglion (DRG) neurons. HGF also enhances the neurotrophic activities of NGF in vitro, and Met receptor signaling is required for the survival of a proportion of DRG neurons in vivo. This synergism is specific for NGF but not for the related neurotrophins BDNF and NT3. By using a mild signaling mutant of Met, we have demonstrated previously that Met requires signaling via the adapter molecule Grb2 to induce proliferation of myoblasts. In contrast, the actions of HGF on sensory neurons are mediated by Met effectors distinct from Grb2. Our findings demonstrate a requirement for Met signaling in neurons during development.     

Electro-acupuncture induced NGF,BDNF and NT-3 expression in spared L6 dorsal root ganglion in cats subjected to removal of adjacent ganglia

This study evaluated the effect of electro-acupuncture (EA) on the NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion (DRG) in cats subjected to bilateral removal of L1-L5 and L7-S2 DRG, using immunostaining, in situ hybridization and RT-PCR. The positive products of NGF, NT-3 protein and mRNA in the small and large neurons of spared L6 DRG in EA side increased greatly more than that of control side, while the increased BDNF was only noted in small and medium-sized neurons. RT-PCR demonstrated that the mRNA level for three factors was not influenced by EA in intact DRG, when a significant increase was seen in the spared L6 DRG of EA side. As it has been well known that DRG neurons project to the spinal cord wherein morphological plasticity has been present after DRG removal, the present results might have some bearing to the observed phenomenon.     

Formation of a full complement of cranial proprioceptors requires multiple neurotrophins.

Inactivation of neurotrophin-3 (NT3) completely blocks the development of limb proprioceptive neurons and their end organs, the muscle spindles. We examined whether cranial proprioceptive neurons of the trigeminal mesencephalic nucleus (TMN) require NT3, brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT4) for their development. Complements of TMN neurons and masticatory muscle spindles were decreased by 62% in NT3 null mutants, 33% in BDNF null mutants, and 10% in NT4 null mutant mice at birth. The extent of proprioceptive deficiencies differed among different masticatory muscles, particularly in NT3 null mice. Masticatory muscles of embryonic mice heterozygous for the NT3(lacZneo) or BDNF(lacZ) reporter genes expressed both NT3 and BDNF, consistent with target-derived neurotrophin support of TMN neurons. Although more than 90% of TMN neurons expressed TrkB as well as TrkC receptor proteins by immunocytochemistry in wild-type newborns, TrkC or TrkB null mice exhibited only partial proprioceptive deficiencies similar to those present in NT3 or BDNF;NT4 null mice. Thus, in terms of the survival outcome, two main subpopulations of TMN neurons may exist during embryogenesis, one dependent on TrkC/NT3 functioning and the other utilizing TrkB/BDNF signaling. The differential dependence of TMN neurons on neurotrophins may reflect differential accessibility of the neurons to limiting amounts of NT3, BDNF, or NT4 in target tissues, especially if the tissue distribution or levels of BDNF, NT3, and NT4 were dynamically regulated both spatially and temporally.     

Early postnatal corticosterone administration regulates neurotrophins and their receptors in septum and hippocampus of the rat

The principal glucocorticoid in rats, corticosterone, interacts with neurons in the limbic system and leads to morphological and behavioral changes. Putative corticosterone-triggered mediators are neurotrophins. In the present study we investigated the effects of early postnatal corticosterone treatment in rats on neurotrophic factors of the nerve growth factor (NGF) family and their receptors. Newborn rats were treated with corticosterone-containing polymers until postnatal day 12. The mRNA and protein levels of the neurotrophins of the NGF family (NGF, BDNF, NT-3 and NT-4/5) and their receptors (trkA, trkB, trkC and p75) were quantified in septum and hippocampus using RT-PCR. In the septal region, we found an unchanged mRNA expression after corticosterone treatment, whereas in the hippocampus there was a general increase in mRNA. Particularly, the gene expression of NGF, NT-3, and the high affinity receptors trkA, trkB and trkC increased significantly. Quantification of the neurotrophin protein levels using an ELISA revealed significant treatment effects for NGF and NT-4/5 in the hippocampus. The present study of corticosterone treatment in young rats demonstrates interactions of steroid hormones with neurotrophic factors and their receptors in the septo-hippocampal system during the first two postnatal weeks.