成年SD大鼠的NGF药代动力学研究

采取静脉注射和皮下注射两种给药方式,对清洁级成成年ＳＤ大鼠分别于给药后不同时间采取尾静脉血,通过双抗体夹心型ＥＬＩＳＡ方法检测了血清ＮＧＦ的含量,并求出了得主要药代动力学参数。     

人神经生长因子的规模纯化

建立一种简便、快捷的人神经生长因子（ｈｕｍａｎ Ｎｅｒｖｅ Ｇｔｏｗｔｈ Ｆａｃｔｏｒ, ｈＮＧＦ）的规模纯化方法,获得批量的ｈＮＧＦ。方法：以胎盘为原料,根据ｈＮＧＦ的性质,采用匀浆、超滤及一步层析法,规模纯化ｈＮＧＦ,并用ＳＤＳ－ＰＡＧＥ及等电聚焦分别测定其分子量和等电点,利用鸡胚背根神经节法检测ｈＮＧＦ的生物活性。结果;纯化得单一组分的ｈＮＧＦ,测得其分子量约１４．４ ｋＤ,等电点约为９．３,比活约１．５×１０~４Ｕ／ｍｇ。纯化产物与抗重组人神经生长因子抗体有很强的免疫交叉反应。结论：该纯化工艺可得到批量、高纯度的人神经生长因子。     

pcDNA3-hNGFb真核表达载体的构建及其表达

目的构建人神经生长因子β（NGF-β）基因真核表达载体并观察其在L929细胞内的表达。方法以RT-PCR从人脑组织总RNA中扩增NGF-βcDNA,将其克隆到真核表达载体pcD-NA3中,经酶切鉴定和序列分析后,以Lipofectamine2000介导转染L929细胞,应用免疫细胞化学和western blot鉴定NGF-β在细胞内的表达。结果RT-PCR产物为750bp的特异片段,重组质粒pcDNA3-hNGFb酶切后产生750bp和5.2kb的片段,DNA测序证实750bp片段的碱基序列与人NGF-βcDNA完全一致。将其转染L929细胞后,免疫细胞化学、western blot结果表明NGF-β及其前体proNGF-β能在真核细胞中正确表达。结论成功构建了重组真核表达质粒pcDNA3-hNGFb,为后续的研究奠定基础。     

Cholinergic but not monoaminergic denervation increases nerve growth factor content in the adult rat hippocampus and cerebral cortex

Summary We have investigated whether degeneration of basal forebrain cholinergic neurons is a potential trigger for increased NGF production in the adult rat brain. Electrolytic lesions of cholinergic neurons in the septum-diagonal band and in the nucleus basalis of Meynert induced a transient increase in NGF in the ventral hippocampus (+70%) and cerebral cortex (+125%), respectively. In contrast, selective aminergic denervation of the forebrain by electrolytic lesion of the medial forebrain bundle, did not increase NGF levels in hippocampus and cerebral cortex. Thus, a cholinergic mechanism appears to regulate NGF production in adult rat basal forebrain.     

Development of neuronal markers in aggregating fetal rat telencephalon cells cultured in the presence of triiodothyronine

The concentrations of the general neuronal markers D2-protein (N-CAM), D3-protein and neuron specific enolase (NSE) in reaggregating cultures of fetal rat telencephalon cells were affected by the presence of 30 nM triiodothyronine in the defined culture medium. The extent of normal developmental changes were enhanced by triiodothyronine, as demonstrated by crossed immunoelectrophoresis. From 13 to 19 days in culture, the concentration of D2-protein decreased, and the concentrations of both D3-protein and NSE increased. Nerve growth factor (NGF) was without effect on the development of these general neuronal markers. However, as shown previously both triiodothyronine and NGF increased the activity of choline acetyltransferase, a marker for cholinergic neurons. The results suggest an enhanced overall differentiation of several types of telencephalon neurons in the presence of triiodothyronine, and a specific stimulation of cholinergic telencephalon neurons by NGF.     

Selective induction of nerve growth factor and brain-derived neurotrophic factor by LPS and allergen in dendritic cells

Background Neurotrophins are produced by various cells upon different stimuli and participate in the initiation and regulation of inflammation in various diseases including allergy and asthma, but little is known about the production and control of neurotrophins by dendritic cells (DCs). The aim of this study was to assess whether DCs produce the neurotrophins nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF), and whether inflammatory stimuli or allergens are able to induce the production of neurotrophic factors. Methods Monocyte‐derived dendritic cells (MoDCs) were generated from different donors. The neurotrophins NGF and BDNF were demonstrated by RT‐PCR, Western blotting, flow cytometry analysis and fluorescence microscopy. MoDCs were cultured and stimulated with lipopolysaccharide (LPS) or allergen for 24 h. The supernatants and cells were collected. Measurement for NGF and BDNF was performed by ELISA. Results DCs express mRNA for the neurotrophins NGF and BDNF. Proteins were detectable by Western blot, FACS analysis and fluorescence microscopy. LPS led to an up‐regulation of BDNF, while NGF was unaffected. Cell lysates demonstrated an increased amount of BDNF after stimulation with LPS or allergen, while NGF was not affected significantly. Conclusions DCs are a source of neurotrophins. LPS selectively regulates the production of BDNF. Allergen stimulation leads to an LPS‐independent regulation. This contributes to a complex involvement of neurotrophins in allergic diseases.     

Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. II. A conditioning lesion paradigm

The PC12 rat pheochromocytoma cell line is a nerve growth factor (NGF) responsive line that is protected by NGF from the toxic effects of hydrogen peroxide induced peroxidation. In part, NGF protection of PC12 cells acts through a shift in oxidant-antioxidant metabolism by the enhancement of catalase activity. When PC12 cells are used in a conditioning lesion paradigm to study the effects of an initial sublethal peroxidative insult on subsequent responses to injury, a low dose conditioning lesion protects even in the absence of NGF. The magnitude of the protective effect exerted by the conditioning lesion, however, is augmented in the presence of NGF, since a significant cytoprotective effect is observed over a wider range of H2O2 concentrations. Neuronal injury due to treatment with a high dose of H2O2 (5 mM) has a cytotoxic effect that cannot be prevented by NGF treatment and is, in itself, not conditioning in nature. This in vitro model system lends itself to the development of explanations regarding the salutory effects of conditioning lesions at the molecular level.     

Reduced serum concentrations of nerve growth factor, but not brain-derived neurotrophic factor, in chronic cannabis abusers

Chronic cannabis use produces effects within the central nervous system (CNS) which include deficits in learning and attention tasks and decreased brain volume. Neurotrophins, in particular nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), are proteins that serve as survival factors for CNS neurons. Deficits in the production and utilization of these proteins can lead to CNS dysfunctions including those associated with cannabis abuse.In this study we measured by enzyme-linked immunosorbent assay (ELISA) the NGF and BDNF serum levels in two groups of subjects: cannabis-dependent patients and healthy subjects. We found that NGF serum levels were significantly reduced in cannabis abusers as compared to healthy subjects.These findings indicate that NGF may have a role in the central action of cannabis and potentially in the neurotoxicity induced by this drug. These data also suggest that chronic cannabis consumption may be a risk factor for developing psychosis among drug users.