Human mast cells express functional TrkA and are a source of nerve growth factor

Mast cells are the principal effector cells in IgE-dependent hypersensitivity reactions. Despite reports that rodent mast cells proliferate in the presence of nerve growth factor (NGF), human mast cells reportedly do not respond to this factor. To determine if human mast cells express the NGF receptors, TrkA tyrosine receptor and the low affinity NGF receptor (LNGFR), we first analyzed the mRNA expression by RT-PCR of TrkA and LNGFR in a human mast cell line (HMC-1) and in human mast cells cultured in the presence of stem cell factor. Both HMC-1 and cultured human mast cells were found to express TrkA but not LNGFR. TrkA protein was demonstrated by Western blot analysis of HMC-1 lysates. Using flow cytometric analysis and mast cell tryptase as a mast cell marker, both HMC-1 cells and cultured human mast cells were shown to co-express tryptase and TrkA. Treatment of mast cells with NGF resulted in phosphorylation of TrkA on tyrosine residues as detected by immunoblotting with an antiphosphotyrosine antibody. Furthermore, NGF induced the immediate early gene c-fos in HMC-1 cells. HMC-1 cells and cultured human mast cells were also found to express NGF mRNA, and conditioned medium from HMC-1 cells stimulated neurite outgrowth from chicken embryonic sensory ganglia in culture. This effect was blocked by anti-NGF. Thus, mast cells express functional TrkA and synthesize NGF, suggesting a mechanism by which NGF may act as an autocrine factor for human mast cells, and by which mast cells and nerves may interact.     

Immunocytochemical demonstration of nerve growth factor receptor (NGF-R) in developing human fetal teeth

Evidence is accumulating that nerve growth factor receptor (NGF-R or p75^NGFR) can mediate cell growth and differentiation of non-neuronal cells. NGF-R expression was studied in developing teeth of human embryos and fetuses between the 6th and 18th weeks of gestation, using a monoclonal mouse-anti-human NGFR antibody. In contrast to earlier findings in rodents, the NGF-R expression of the human dental papilla was found to be transient. NGF-R was present in the condensing ecto-mesenchymal cells of the dental papilla in the early cap stage tooth germ. In later developmental stages, a shift of the NGF-R expression from the papilla to the cytoplasmic membrane of the inner enamel epithelium (IEE) was demonstrated. As in rodent odontogenesis, the NGF-R immunoreactivity of the IEE remained until the odontoblasts started secretion of predentinal matrix in the late bell state. The mitotic activity in the IEE was detected by an antibody against proliferating cell nuclear antigen (PCNA) and showed that the NGFR expression of the IEE decreased as the cell proliferation ceased. We propose that NGF-R may, be involved in differentiational and/or proliferative events of human odontogenesis.     

神经生长因子在糖尿病足创面组织中表达相关性的研究进展

动物实验研究证实神经生长因子（NGF）促进创面组织血管生成、血管内皮细胞再生,促进缺血创面的愈合及糖尿病创面的愈合。通过NGF在糖尿病创面表达改变的研究,有助于NGF促进糖尿病创面愈合机制的探讨,为糖尿病足创面愈合提供新的理论依据。     

异硫氰酸荧光素标记的神经生长因子缓释微球的制备及评价

目的 探讨异硫氰酸荧光素(FITC)标记的神经生长因子缓释微球的制备,并对其进行体内外评价.方法 采用水-油-水的双乳化技术制备FITC标记的神经生长因子缓释微球.利用扫描电镜和荧光显微镜对其形态特征进行观察,并对其体内外释放情况进行研究.结果 制备的FITC标记的神经生长因子缓释微球包封率和载药量分别为(97.9±8.9)%和(4.90±0.56)%.扫描电镜结果显示所制备的微球呈圆形、形态规整、粒径分布较均匀.荧光显微镜结果显示所包载的蛋白类药物在微球内旱随机分布.缓释微球体外持续释放5周后,有73%的蛋白释放出来;荧光示踪显示在体内能够持续释放达5周以上.结论 采用水-油-水的双乳化技术制备的缓释微球可以将生物大分子药物如神经生长因子成功运载到脑内.     

载NGF海藻酸凝胶-聚乳酸复合导管修复兔面神经缺损实验研究

目的    观察载神经生长因子（nerve growth factors, NGF）海藻酸凝胶-聚乳酸复合导管修复面神经缺损的作用。  方法    建立白兔面神经缺损模型,将面神经断端植入制备好的载NGF海藻酸凝胶-聚乳酸复合导管内2 mm,使面神经两断端之间距离为10 mm,将导管两端与神经固定缝合皮肤。再随机将12只大白兔分为术后4、8、12周组。实验结束后切开大白兔面神经吻合部位,选取神经导管近、中和远端组织进行HE染色,观察神经生长及导管降解情况。  结果    实验白兔手术伤口Ⅰ期愈合,无局部坏死、移植物排出及全身不良反应。术后4周导管壁有巨噬细胞浸润,导管近端雪旺细胞、成纤维细胞增生,新生小血管丰富;8周可见导管壁变薄,有成纤维细胞附着,巨噬细胞浸润,导管内有神经束样结构,束间纤维分隔,外层纤维包膜增厚,新生小血管减少;12周可见神经导管壁进一步分解,胶原纤维增生,导管内神经纤维结构接近正常,轴突样结构增粗,外层纤维包膜变薄。  结论    载NGF海藻酸凝胶-聚乳酸复合导管能较好的引导面神经再生,具有良好的组织相容性。     

Effects of nerve growth factor and glucocorticoid on cultured human pheochromocytoma cells

Primary cell cultures of two human pheochromocytomas (PC) that were associated with high serum levels of adrenaline and noradrenaline were developed to study the effects of nerve growth factor (NGF) and dexamethasone on the morphology and function of PC cells in vitro. By phase-contrast microscopy, cultured cells were small and hyperchromatic on the first day of culture; neurite-like processes that extended to other cells developed several days later and were maintained for more than 3 months. NGF (100ng/ml), dexamethasone (10^–5M), or NGF + dexamethasone were added to the culture media 2 weeks after the cultured cells had stabilized. Catecholamine concentrations in the medium were maintained at higher levels after addition of NGF, dexamethasone, or NGF + dexamethasone as compared to control cells. In the presence of NGF, extension of neurite-like processes was clearly accelerated, while high levels of dexamethasone inhibited growth of processes. These in vitro studies showed that the addition of NGF or the removal of dexamethasone induces differentiation of adrenal neurons present in pheochromocytomas, suggesting that adrenocortical steroid hormones influence the morphological control of adrenal medullary cells.     

Frequent expression of 75 kDa nerve growth factor receptor and phosphotyrosine in human peripheral nerve tumours: an immunohistochemical study on paraffin-embedded tissues

One hundred and three benign, and 10 malignant peripheral nerve tumours were examined immunohistochemically for expression of 75 kDa nerve growth factor receptor (NGFR). In benign tumours NGFR was demonstrated at 61% in neurinoma, 71% in neurofibroma, 93% in neurofibromatosis and 90% in traumatic neuroma. Malignant neurogenic tumours were 100% positive for NGFR. Phosphotyrosine-immunoreactivity was detected in 76% of NGFR-positive tumours but the frequency of immunostained tumour cells was low. These results suggest that both benign and malignant peripheral nerve tumours express 75 kDa NGFR. The receptor seems to serve as growth signal transduction of the tumour cells in terms of phosphorylation of the tyrosine residue of the receptor or the target protein of the NGFR protein tyrosine kinase.     

神经生长因子在糖尿病足创面组织中的表达

目的研究神经生长因子(NGF)及其高亲和性的酪氨酸激酶-A受体(TrkA)在糖尿病足创面组织中的表达。方法 收集糖尿病足创面组织标本作为对照组,正常人皮肤及皮下软组织标本作为正常组,采用免疫组化的方法观察神经生长因子(NGF)及其高亲和性的TrkA受体的表达,酶联免疫吸附测定法(ELISA)测定NGF的含量。结果 对照组NGF含量为(66.299±10.204)pg/ml,与正常组NGF含量(35.015±4.671)pg/ml相比,差异有统计学意义(P=0.010)。结论 NGF及其高亲和性的TrkA受体在糖尿病足创面组织中表达的变化可能参与了创面的发生发展,是糖尿病足创面修复的影响因素之一。     

寻常型银屑病患者皮损中瘦素、脂联素与血管内皮生长因子表达的研究

目的 探讨寻常型银屑病患者皮损中瘦素、脂联素及其血管内皮生长因子vascular endothelial growth factor(VEGF)的变化与意义.方法 ①采用免疫组织化学技术检测34例寻常型银屑病患者和21例正常人皮损中瘦素、脂联素与VEGF表达水平.结果 同正常对照组相比,寻常型银屑病患者皮损中瘦素、脂联素以及血管内皮生长因子表达明显高于正常对照组,强阳性率分别为17.65%、17.65%和14.7%,差异有统计学意义(χ2=9.18,P<0.05;χ2=13.34,P<0.05;χ2=18.9,P<0.05),相关分析表明,瘦素表达水平与VEGF表达水平呈正相关(r=0.8,P<0.05),脂联素与VEGF无相关(r=-0.02,P>0.05).结论 寻常型银屑病患者皮损中瘦素、脂联素及其VEGF水平的变化可能与银屑病的发病有关,而血管内皮生长因子的变化可能与瘦素有关.     

Low-frequency electro-acupuncture reduces the nociceptive response and the pain mediator enhancement induced by nerve growth factor

A number of studies have shown that the potential clinical benefits of nerve growth factor (NGF) administration are limited by its hyperalgesic side effects. The ancient therapeutic technique of acupuncture and its modern derivate electro-acupuncture (EA) have been proven effective in reducing hyperalgesia as well as nociceptive and neuropathic pain in several pathological conditions. The present study addresses the question of whether EA can influence the hyperalgesia induced by NGF administration. We treated adult healthy rats with repeated injections of murine NGF and/or low-frequency electro-acupuncture. We found that EA was able to counteract the NGF-induced hyperalgesic response when assessed by a hot plate test. Moreover, EA counteracted the NGF-driven variation of substance P (SP) and transient receptor potential vanilloid type 1 (TRPV1) response in both hind-paw skin as well as the corresponding dorsal root ganglia (DRG). Our findings indicate that low-frequency EA could be useful as a supportive therapy to reduce NGF-induced side effects, such as hypersensitivity and hyperalgesia, when clinical treatment with NGF is necessary.     

Brain derived neurotrophic factor is increased in cerebrospinal fluid of children suffering from asphyxia

Brain derived neurotrophic factor (BDNF) is a neurotrophic factor that is relatively highly expressed in developing and adult brain. Whereas clinical determinations of nerve growth factor (NGF) in human serum and cerebrospinal fluid (CSF) in different conditions have been undertaken there are no reports on levels of BDNF in human CSF. Here we show that BDNF is increased in CSF of neonatal children suffering from asphyxia which is characterised by periods of brain hypoxic-ischemia. In contrast to BDNF, levels of CSF NGF were largely decreased in these children. The present results show that BDNF can be detected in human CSF and that the levels increase following hypoxic-ischemic brain injury. As suggested by animal studies the increased BDNF might counteract neuronal damage observed in these patients following asphyxia.     

The activated nerve growth factor receptor p-TrkA is selectively expressed in advanced-stage ovarian carcinoma

The objective of this study was to compare the expression of the nerve growth factor (NGF) receptors TrkA and p75 in ovarian borderline tumors, International Federation of Gynecology and Obstetrics (FIGO) stage I carcinomas and advanced-stage (FIGO stage III-IV) carcinomas, and to assess a possible association between NGF receptor expression and mitogen-activated protein kinase (MAPK) activation in borderline tumors and FIGO stage I carcinomas. Sections from 119 borderline tumors, 57 FIGO stage I invasive ovarian carcinomas, and 56 advanced-stage carcinomas were evaluated for expression of activated phospho-TrkA (p-TrkA) and p75 using immunohistochemistry. MAPK activation was analyzed in stage I carcinomas and borderline tumors using phospho-specific antibodies against the extracellular-regulated kinase (p-ERK), the high osmolarity glycerol response kinase (p-p38), and the c-jun amino-terminal kinase (p-JNK). p-TrkA membrane expression was significantly more frequent in advanced-stage carcinomas compared with both borderline and stage I carcinomas (P < .001). p75 membrane expression was comparable in the 3 groups (P > .05). p-ERK and p-p38 expression was comparable in borderline and stage I carcinomas, whereas p-JNK was more frequently expressed in stage I ovarian carcinomas (P < .001). NGF receptor expression showed no association with MAPK activation in borderline and stage I carcinomas. In conclusion, expression of biologically active p-TrkA receptor at the cell membrane is up-regulated along tumor progression in ovarian carcinoma, whereas p75 expression remains unaltered. These data provide further evidence regarding the clinical role of p-TrkA in ovarian carcinoma. NGF receptors probably signal via MAPK-independent pathways in ovarian carcinoma.     

鼠神经生长因子治疗胫骨骨不连效果的实验研究

目的　研究鼠神经生长因子（NGF）对白兔胫骨骨不连的治疗效果,为NGF在骨不连的临床运用提供实验基础。 方法　选择健康成年雄性新西兰大白兔40只,随机各选20只分为实验组和对照组,分别于右胫骨粗隆下3~5 cm处造成横行骨折骨不连模型,实验组在骨折处每周注射NGF一次,连续12周;对照组在骨折处每周注射生理盐水一次,连续12周。试验期间两组均同时接受X线和组织学检查以观察骨痂生长和骨折愈合情况。 结果　实验组动物骨痂出现时间为（2.0±0.5）周,骨折线模糊时间为（8.2±1.2）周,骨折愈合时间为（12±1.7）周;对照组骨痂出现时间为8周以上,未出现骨折线模糊和骨折愈合。实验组骨折愈合情况远好于对照组,差异具有统计学意义（P<0.05）。 结论　NGF有较强的促进骨不连骨痂生长和愈合作用,NGF在骨不连的临床治疗中有良好的运用前景。     

Nerve growth factor signal transduction in human B lymphocytes is mediated by gp140trk

Nerve growth factor (NGF) plays an important role in the regulation of the immune system. Recent studies from this laboratory demonstrated the presence of functional NGF receptors on human B lymphocytes; in addition, NGF has been shown to enhance B lymphocyte proliferation. NGF caused both concentration- and time-dependent increases in tyrosine phosphorylation of five proteins of 140, 110, 85, 60 and 42 kDa, which were identified as phospholipase C-γ1, phosphatidylinositol-3 kinase and mitogen-activated protein kinase. To elucidate the contribution of the Trk family of tyrosine kinases to the phosphorylation events induced by NGF, we identified gp140^trk in human B cells and in human B cell lines. Analysis of specific gp140^trk immunoprecipitates indicated that addition of NGF to B cells induced a rapid increase in the tyrosine phosphorylation of gp140^trk and inhibition of this phosphorylation prevented the tyrosine phosphorylation of other proteins. These data identify the central role of gp140^trk in NGF signaling of human B lymphocytes.     

Bilateral phasic increases in dorsal root ganglia nerve growth factor synthesis after unilateral sciatic nerve crush

The amount of nerve growth factor (NGF) in the L5, L6, and cervical dorsal root ganglia of rats was examined from 1 to 30 days after a unilateral crush lesion of the sciatic nerve and adjacent branches of the lumbar plexus at the level of the sciatic notch. Unilateral nerve crush produced increases in NGF content of lumbar ganglia at 1, 4, and 7–8 days after injury, with increased NGF mRNA at 4 and 7–8 days. Increases in NGF at 1 and 4 days were most pronounced on the unlesioned side while increases at days 7 and 8 were most pronounced on the lesioned side. NGF content increased in cervical ganglia of nerve-lesioned animals at 3 and 7 days after injury and in lumbar and cervical ganglia of sham-operated animals 3–5 days after surgery, with no comparable changes in NGF mRNA. Elevations of ganglionic NGF coincide temporally with some of the alterations in metabolism and morphology which occur in dorsal root ganglion neurons after sciatic nerve crush. However, the bilateral nature of increases in NGF demonstrates that the factor(s) producing the response is not restricted to ganglia axotomized by the injury. The data suggest that ganglionic NGF may be regulated by systemic factors, produced during stress or trauma, as well as by factors from the denervated target tissue and/or regenerating axons.     

Temporal pattern of nerve growth factor receptor expression in developing cochlear and vestibular ganglia in quail and mouse

Summary We have previously demonstrated the presence of specific receptors for nerve growth factor (NGF) in cochleovestibular ganglia of 72 h (stage 19–20) quail embryos, with a greater density of NGF receptors in the cochlear portion of the ganglion. The present study was conducted to determine the temporal pattern of NGF receptor expression in cochlear and vestibular ganglia throughout development, and was conducted in two species, quail and mouse. As in the quail, specific binding of¹²⁵I-NGF was detected in cochleovestibular ganglia of mouse embryos from an embryonic age equivalent to 72 h quail embryos (embryonic day 11, E11), with a similar concentration of¹²⁵I-NGF binding in the cochlear portion. Quantitative studies revealed that¹²⁵I-NGF binding continued to increase, in both cochlear and vestibular ganglia, for several days of development, and then began to decrease to minimal levels. Maximal levels were achieved at E7 in the quail, and E14 to E16 in the mouse, while minimal levels were reached by E13 in the quail, and E18 in the mouse. The level of¹²⁵I-NGF binding in cochlear ganglia was two to three times higher than in vestibular ganglia; a finding corroborated by radioautographic studies. In both quail and mouse, NGF receptors were more heavily concentrated in the ventromedial portion of the cochlear ganglion, adjacent to the cochlear duct; an area containing both support cells and peripheral neuronal processes. In the vestibular ganglion,¹²⁵I-NGF binding was more homogeneous, although small areas containing high densities of silver grains were observed. The presence of NGF receptors in cochlear and vestibular ganglia suggests that these ganglia may be responsive to and/or dependent upon NGF during their development.     

Developmental changes in nerve growth factor (NGF) binding and NGF receptor proteins trkA and p75 in the facial nerve

This study characterizes the temporal-spatial distribution of nerve growth factor (NGF) low (p75) and high-affinity (trkA) receptors in the facial nerve and geniculate ganglion (GG) of developing quail embryos (E-3 to E-14). We used ¹²⁵I-labeled NGF (¹²⁵I-NGF) to study binding dynamics in a temporal series of isolated primordia and an autoradiographic series of staged specimens to characterize the occurrence and distribution of NGF receptors in this cranial nerve and its ganglion. In addition, expression of trkA and p75 protein-like immunoreactivity in the facial nerve and GG was studied by Western blot, in order to distinguish between high- and low-affinity NGF receptors respectively. The quantitative study of binding show that isolated facial primordia ranging from E-3 to E-14 exhibit different levels of specific binding. High initial binding levels were observed on E-3 specimens, then an initial decrease on day 4 (E-4) followed by a steady increase from days E-4 to E-7. Maximum ¹²⁵I-NGF binding was achieved on E-7, followed by a steady decline in binding on days 8 (E-8) and 9 (E-9), reaching near background levels on day 10 (E-10) of development and until the oldest stage assayed (E-14). Most of the cells bearing NGF receptors appeared to be nonneuronal crest-derived cells, but some placode-derived neurons and motor fibers of the VIIth cranial nerve transiently expressed the ability to bind ¹²⁵I-NGF. The temporal pattern of p75 expression matches the pattern of quantitative binding of NGF, while the trkA expression is restricted to a few stages mainly E7 and E9, implying that most of the binding detected is via low-affinity receptors, except for a proportion of high-affinity receptors present at stages of maximum binding. This temporal pattern of NGF binding sites suggests that cells within the VIIth cranial nerve are responsive to and/or dependent upon NGF in vivo, so NGF may play a biological role during normal development of the facial nerve. In view of the developmental events that parallel the occurrence and type of NGF binding sites, we suggest that this role may be to modulate from earlier chemotaxis and cell proliferation to much later events, such as neuronal differentiation and neuron-glia interactions. The significance of these findings in regeneration during adult life remain to be investigated.     

神经生长因子对大鼠跨区穿支皮瓣成活的影响

目的　评价神经生长因子（NGF）对SD大鼠背部跨区穿支皮瓣成活的影响。 方法　成年SD大鼠60只,平均分为实验组和对照组。切取面积约3cm×10cm的背部跨区皮瓣。实验组皮下注射NGF溶液（10 nmol·ml-1·kg-1）;对照组同法注射0.1 mol/l磷酸盐缓冲溶液（1 ml/kg）。术后第3、7天局部取材运用Western Blot技术半定量检测VEGF及CD34蛋白含量;显微CT微血管三维重建,析因设计分析血管容积及总长度的形态学变化。后术第7天统计皮瓣存活面积,免疫组化染色观测血管生长因子受体(KDR)及NGF受体(TrkA)的表达。 结果　第3天,VEGF表达实验组与对照组无显著统计学差异（P=0.088）;实验组CD34表达明显高于对照组(P=0.004);第7天,两种蛋白分子表达,实验组显著均优于对照组(P<0.05)。血管形态学分析示NGF和时间因相互作用增加血管容积（F=33.304,P<0.05）及总长度（F=8.493,P=0.01）;实验组皮瓣成活面积明显大于对照组（P<0.05）。免疫组化染色示实验组KDR阳性表达较为明显。 结论　NGF可促进跨区供血穿支皮瓣的成活。     

神经生长因子促进海马胆碱能神经再生过程中Lhx8的表达

目的 探讨神经生长因子（NGF）促进海马胆碱能神经再生与同源盒基因Lhx8之间的关系。方法 SD大鼠72只,在制备切割右侧穹隆海马伞模型和NGF侧脑室注射的基础上,分为对照组、切割组、NGF组、切割联合NGF组;Real-time PCR和Western blotting分别检测各组动物海马中Lhx8蛋白的表达变化;免疫荧光技术检测各组动物海马齿状回颗粒下层中胆碱乙酰转移酶(ChAT)/Lhx8双标细胞数。 结果 切割组和NGF组中Lhx8蛋白的表达量较对照组增高,切割联合NGF组增高更为明显,且Lhx8基因表达也增高;NGF组和切割联合NGF组中海马齿状回颗粒下层(SGZ)中的ChAT/Lhx8双标细胞数也较对照组和切割组明显增多。结论 侧脑室注射NGF促进海马胆碱能神经再生与Lhx8的高表达有关。