中华眼镜蛇毒神经生长因子对鸡胚背根神经节及PC12细胞作用的观察

目的 采用国际上常用的两种测定神经生长因子活性方法对只结眼镜蛇毒神经生长因子活性作用进行观察。方法 鸡胚背根神经节培养法，ＰＣ１２细胞２法。结果 鸡胚背根节培养法在样品达５０ｎｇ／ｍｌ时有活性反应，ＰＣ１２细胞培养法在定笥测定，样品浓度为２０ｎｇ／ｍｌ时能观察到活性，在用定量测定方法时，实验组细胞聚团并长出神经状纤维，说明有活性。     

2种测定神经生长因子活性方法的比较研究

目的：比较２种测定神经生长因子活性的最常用方法，对活性测定条件进行了部分改进。方法：鸡胚背根神经节组织培养法，ＰＣ１２细胞培养法。结果：鸡胚背根神经节培养法灵敏度为５０μｇ．Ｌ＾－１。ＰＣ１２细胞用于定性测定灵敏度为２０μｇ．Ｌ＾－１。ＰＣ１２细胞用于定量测定灵敏度可达１μｇ．Ｌ＾－１。结论：鸡胚背根神经节培养法实验条件要求简单，易于开展，但操作复杂，干扰因素多，一般用于定性测定。ＲＣ１２细胞培养     

广西眼镜蛇蛇毒神经生长因子在大肠杆菌中的表达

目的采用基因工程技术,在大肠杆菌(E.coli)中表达广西眼镜蛇(黑眼镜蛇)蛇毒神经生长因子(NGF)成熟蛋白并检测其生物活性。方法将天然广西眼镜蛇蛇毒的NGF蛋白基因克隆至融合表达载体pET-40b(+)中,以C端融合了6个组氨酸的形式在E.coli BL21(DE3)通过异丙基硫代半乳糖苷诱导表达NGF蛋白。超声破菌后,将上清液经Ni~(2+)-NTA柱纯化,收集并冻干NGF融合蛋白,用蛋白印迹法分析其NGF抗原活性,并通过促PC12细胞生长法观察其生物活性。结果经蛋白印迹分析可知在38 ku处的条带具有NGF抗原活性。加入天然蛇毒NGF,生长轴突的PC12细胞为(83±3)%,重组品为(21±3)%,表明经纯化的融合蛋白具有NGF生物活力,但活性弱于天然蛇毒NGF。结论在大肠杆菌中可以表达出有活性的蛇毒NGF蛋白。     

N-芳磺酰基α-烷基L-氨基酸酯的合成及其神经营养活性

目的设计合成N－芳磺酰基α－烷基L－氨基酸酯化合物，并对其神经营养活性进行初步评价。方法 基于先期工作，以生物电子等排原理，设计合成系列N－芳磺酰基α－烷基L－氨基酸酯化合物，并运用体外PC12细胞存活模型和鸡胚背根神经节无血清培养方法，观察化合物的神经营养作用。结果 共合成32个目标化合物，其结构均经^1H-NMR和MS确证。结论 化合物3，5，10，12，13，14，15，20，21和28可明显增强神经生长因子（NGF）促PC12细胞体外存活活性；化合物12，13，15，20和28同时具有较强的促鸡胚背根神经节突起生长作用，其中化合物12和15活性突出，优于阳性对照化合物GPI－1046。     

江浙蝮蛇蛇毒中神经生长因子的分离新方法探讨

为从江浙蝮蛇蛇毒中分离出神经生长因子,采用 CM-Sepharose FF 及 Sephacryl S-200柱层析方法进行了分离纯化,从中分离出一种组分。该组分经鸡胚背根神经体外培养证明具有促进神经节神经纤维生长的活性,证实为神经生长因子。经聚丙烯酰胺凝胶电泳检测为一条区带,其分子量约为27000,该试验结果表明用此种方法从江浙蝮蛇中提取神经生长因子是简便可行的。     

Cobra Venom NGF Inhibits Proliferation and Induces Apoptosis of Hepatic Stellate Cell

目的:从眼镜蛇毒中分离纯化神经生长因子(Nerve Growth Factor,NGF),观察眼镜NGF对肝星状细胞HSC-T6增殖、调亡活性的影响,进一步为蛇毒NGF在抗肝纤维化治疗提供依据.方法:采用shephadex G-75和CM Sepharose CL-6B二步柱色谱对眼镜蛇毒NGF进行纯化分离;PC12细胞测定各洗脱峰的活性,再用SDS-PAGE鉴定具有NGF活性洗脱峰的纯度和相对分子质量.实验设立空白对照和NGF处理组,分别作用于HSC-T6,培育相应时间,MTT检测眼镜蛇毒NGF对HSC-T6细胞活力影响;HE染色、紫外激光显微镜与透射电镜观察HSC-T6细胞的形态学变化;TUNEL、流式细胞技术检测眼镜蛇毒NGF对HSC-T6细胞凋亡的影响.结果:眼镜蛇毒经PC-12细胞鉴定第Ⅵ峰具有NGF活性;SDS-PAGE检测为电泳纯,相对分子质量为22.3KD;NGF对HSC-T6细胞增殖具有明显抑制作用(2μg/ml NGF的抑制率为49.66％±6.50％,P＜0.05;6.25 μg/ml NGF的抑制率为71.33％±1.53％,P＜0.05);TUNEL法检测发现NGF干预组的凋亡率28.71％±1.59％(2ug/ml NGF)和34.4％±2.49％(5 μg/mlNGF)明显高于对照组的15.85％±1.58％(P＜0.05);流式细胞仪也有同样的发现,NGF干预组的凋亡率16.12％±3.02％(2 ug/mlNGF)和21.15％±3.31％(5 μg/ml NGF)明显高于对照组的2.7％±1.55％( P＜0.05).结论:眼镜蛇毒NGF能抑制肝星状细胞HSC-T6增殖并诱导其凋亡.     

小鼠βNGF抗体纯化和活性鉴定

本文报道以小鼠βNGF为抗原免疫家兔，制备βNGF抗血清，用于Western印迹法，测定结果βNGF在分子量约14kDa处显一条带，亲和层析纯化抗血清得到βNGF抗体。采用鸡胚背根神经节培养法测定了βNGF及其抗体的生物学活性。     

蛇毒神经生长因子的进一步纯化与鉴定

对分离的蛇毒神经生长因子进一步纯化,并签定了其部分性质.结果纯化的蛇毒神经生长因子在浓度为5 ng/ml时仍有良好的生物活性。经SDS-PAGE分析,呈电泳一条带,其分子量约为14kD.等电聚焦电泳分析,等电点为6.80.紫外光谱分析,在220.6nm处有一个特异吸收峰,氨基酸组分分析表明由231个18种氨基酸组成。     

一条新的可诱导PC12细胞转化为交感神经原表型的小分子肽链

目的根据神经生长因子 (NGF)的氨基酸序列及其晶体构象资料 ,对 β NGF进行限制性酶解 ,从而获得关键的功能区域片段。方法选择溴化氰在 9位Met处 ,Trypsin在Arg或Lys处裂解 β NGF ,用SephadexG 5 0色谱、DE5 2离子交换色谱和反相高效液相色谱进行分离纯化。所得片段用PC12细胞测定活性 ,对活性片段进行氨基酸组成及序列分析。结果从 β NGF裂解片段中获得一可诱导PC12细胞分化的活性片段 ,氨基酸组成及序列分析表明 ,此片段由 16肽GEFSVCDSVSVWVGDK与 14肽HWNSYCTTTHTFVK通过一对二硫键连接而成 ,与 β NGF肽链的10～ 2 5和 75～ 88氨基酸残基序列相对应。生物活性分析表明其最佳作用浓度为 0 .0 0 1～ 0 .1ng/ml。结论此实验获得了 β NGF关键的功能区域片段 ,虽然其空间结构和功能的关系尚需研究探讨 ,但它的成功分离和鉴定为合成或表达高活性小分子神经营养物质奠定了重要的基础     

神经生长因子的生物活性研究

用鸡胚背根培养法和新生大鼠皮层神经元原代分散培养法对制备的神经生长因子的生物活性进行了研究，实验结果证实了该神经生生因子确有促进神经细胞生长的活性，这为神经生长因子尽快过渡到临床，提供了实验依据。     

Purification and Characterization of a Novel Ngf from Chinese Cobra (Naja Naja Atra) Venom

Abstract

Nerve Growth Factor (NGF) was demonstrated to play an important role in the neuron system; it was required for the development, maintenance, and differentiation of neuron. NGF was purified from the venom of Chinese cobra (Naja naja atra) by the gel filtration on a Sephadex G-50 column, followed by ion-exchange DEAE Cellulose D52 and CM Sepharose CL-6B column chromatography, and then by FPLC on Superose 12 column. The purified NGF was shown to be homogeneous in SDS-PAGE. In vitro, it possessed the biological activity on inducing the neurites growth of the cultured dorsal root ganglia of chicken embryos. Its molecular weight was estimated to be about 23,000 D. The isoelectric point was near 9.2. It was a glucoprotein containing 0.15% neutral hexose. We determined its terminal amino acid sequence, N-NVDFNSESTR, C-IIGNA. In this report, we also discussed the difference in characterizations between this NGF and other Elapidae venom NGFs.     

Characterization of nerve growth factors (NGFs) from snake venoms by use of a novel, quantitative bioassay utilizing pheochromocytoma (PC12) cells overexpressing human trkA receptors.

Snake venoms are a very abundant source of nerve growth factors (NGF). NGFs of Elapidae showing 65% sequence homology with mouse or human NGF, while the Viperidae NGF shows N-glycosylation (Asn-21) typical of these mammalian NGFs. Snake NGF-induced neurite outgrowth (neurotropic activity) was measured in the past by using PC12 cell or dorsal root ganglion bioassays.The present study was aimed at comparing, by dose-response experiments, the neurotropic activity of cobra and vipera versus mammalian NGFs, by using a novel bioassay involving PC12 cells genetically engineered to overexpress NGF-trkA receptors of human origin.These cells respond to NGF by differentiation (morphologically expressed as neurite outgrowth) by a process mediated by NGF-trkA receptors. This process was evaluated by two different criteria: (1) elongation of neurites (E), and (2) Percentage of responsive cells (PRC) determined by digital acquisition of data and computer analysis. We found that snake venom NGFs were less potent than mouse NGF, and that cobra NGF was more potent than vipera NGF. These data indicate the following order of NGF activity towards recombinant human trkA receptors: recombinant human NGF>mouse NGF>cobra NGF>vipera NGF. The neurotropic efficacy of these NGFs was found to be similar, reaching 80-90% of maximal activity obtained with all NGF forms. Interestingly, cobra (but not vipera) NGF demonstrated prolonged neurotropic activity compared with mouse NGF.The results of the present study indicate that cobra and vipera venom NGFs represent natural agonists of human trkA-receptor of a lower potency, but of similar efficacy, compared with mammalian NGFs. These compounds are important pharmacological tools to characterize the trkA receptor structure-function relationship, and to develop novel neurotropic drugs.     

神经生长因子(NGF)对兔角膜缘干细胞增殖的影响

目的:探讨神经生长因子(NGF)对兔角膜缘干细胞增殖作用的影响及最佳有效浓度。方法:取成年大耳白兔角膜缘组织,应用组织块消化培养法进行原代培养,取第2代生长状态良好的干细胞,以5×103个/m l细胞浓度接种于96孔细胞培养板中,依次加入不同浓度的NGF,培养48h后,采用M TT法测定细胞增殖情况。结果:10ng/m l、50ng/m l、100ng/m l、200ng/m l不同浓度组的NGF均有促进角膜缘干细胞的增殖作用,与对照组比较差异均有显著性(P<0.01)。1ng/m l组与对照组、50ng/m l组与200ng/m l组比较差异均无显著性(P>0.05)。结论:NGF可有效促进培养角膜缘干细胞的生长,培养的适宜浓度为10~100 ng/m l,为角膜缘干细胞移植患者术后及角膜外伤或溃疡时局部应用NGF提供了理论依据。     

Chronic exposure of sensory neurones to increased levels of nerve growth factor modulates CB1/TRPV1 receptor crosstalk

BACKGROUND: Anandamide (AEA) activates both cannabinoid CB(1) and TRPV1 receptors, which are expressed on cultured dorsal root ganglion neurones. Increased levels of nerve growth factor (NGF) are associated with chronic pain states. EXPERIMENTAL APPROACH: The aim of this study was to compare of the effects of AEA on CB(1) receptor signalling and TRPV1-CB(1) crosstalk in low and high concentrations of NGF, using voltage-clamp electrophysiology and Fura-2 calcium imaging. KEY RESULTS: Chronic exposure to high NGF (200 ng ml(-1)) as compared to low NGF (20 ng ml(-1)) increases the proportion of neurones that exhibit an inward current in response to AEA (1 microM), from 7 to 29%. In contrast, inhibition of voltage-gated calcium currents by AEA is not significantly different in low NGF (33+/-9%, compared to high NGF 28+/-6%). Crosstalk between CB and TRPV1 receptors is modulated by exposure to high NGF. In low NGF, exposure to the CB(1) receptor antagonist, SR141716A, (100 nM) increases the percentage of neurones in which AEA elicits an increase in [Ca(2+)](i), from 10 to 23%. In high NGF, the antagonist does not alter the percentage of responders (33 to 30%). In low NGF, exposure to the CB receptor agonist, WIN55 (1 microM) reduces capsaicin-mediated increases in [Ca(2+)](i) to 28+/-8% of control as compared to an enhancement to 172+/-26% of control observed in high NGF. CONCLUSIONS AND IMPLICATIONS: We conclude that cannabinoid-mediated modulation of TRPV1 receptor activation is altered after exposure to high NGF.     

小鼠颌下腺神经生长因子的提取及活性鉴定的初步研究

对神经生长因子（Nerve Growth Factor,NGF）提取纯化、活性鉴定进行了研究。将小鼠颌下腺组织匀浆、离心,取其上清,在不同pH和离子强度,依次通过两个离子交换纤维素层析柱,制得 2.5 S NGF纯品。其分子量约43.5 kD,3种主要组分的等电点分别为8.85、9.10和9.20。用鸡胚背根培养法证明其具有刺激神经生长的作用。     

Terpenoids with neurite outgrowth-promoting activity from the branches and leaves of Illicium merrillianum

Eighteen terpenoids (1–18) were isolated from Illicium merrillianum. Compound 1 was identified as new compound, and its structure was established by comprehensive spectroscopic analysis and single-crystal X-ray diffraction. All compounds were evaluated for nerve growth factor (NGF)-mediated neurite outgrowth activity using rat pheochromocytoma (PC12) cells as a model system of neuronal differentiation. Compounds 1, 3, 18 showed significant neurite outgrowth-promoting activity in the presence of 20 ng/ml NGF in a dose-dependent manner at concentrations of 1–100 μM after 24-h treatment. Subtle difference of functional groups at C-2 position in hopane-type triterpene resulted in enormous bioactivity difference, compound 1 was neurotrophic but 2 was cytotoxic.