舟山眼镜蛇毒神经毒素的分离纯化及镇痛作用研究

目的从舟山眼镜蛇毒中分离纯化神经毒素,并测定其部分理化性质及镇痛活性。方法应用SP-Sephadex C-50离子交换色谱法分离眼镜蛇毒粗毒,用Sephadex G-50凝胶过滤色谱、CM-Sephadex C-25离子交换色谱纯化神经毒素;SDS-PAGE(Tris-Tricine系统)鉴定纯度;用Meier方法测定毒性;用热板法观察神经毒素的镇痛作用。结果应用SP-Sephadex C-50离子交换柱层析,从舟山眼镜蛇毒中分离得到14个蛋白峰,其中6个组分(NNAV-Ⅶ ~Ⅻ)经鉴定为含有神经毒素组分,将神经毒活性最强的组分XI经Sephadex G-50、CM-Sephadex C-25纯化得神经毒素纯品NTⅪ。该纯品分子量12.3kD,小白鼠静脉注射和腹腔注射的LD50分别为1.9875、2.2217 mg·kg-1。NTXI能显著提高小鼠对热板刺激的痛阈。腹腔注射0.2mg·kg-1NTXI可使小鼠的热板痛阈提高84.35%。NTXI的镇痛作用具有时效性,给药后2h起效,4h作用达峰值。结论舟山眼镜蛇毒中分离得到一个神经毒素纯品NTⅪ,具有镇痛作用。     

中华眼镜蛇神经毒素微球的制备及镇痛作用研究

目的研究中华眼镜蛇神经毒素PLGA微球制备及通过鼻黏膜给药的镇痛作用。方法采用复乳溶剂挥发法制备眼镜蛇神经毒素PLGA微球,采用大鼠甩尾法测定其镇痛效果。结果采用W/O/O方法,成功制备出中华眼镜蛇神经毒素鼻腔给药PLGA微球,微球平均粒径25μm左右,药物包埋率达80%以上。通过大鼠甩尾法显示明显的镇痛效果。结论将中华眼镜蛇神经毒素制成微球制剂鼻腔给药,镇痛作用明显增强。     

中华眼镜蛇神经毒素的活性研究

目的研究中华眼镜蛇神经毒素(NT)的镇痛活性和对离体蛙腹直肌N2-AChR的拮抗作用。方法采用三种模型(电刺激-嘶叫法,热板法,醋酸扭体法)观察NT的镇痛活性,并观察NT对ACh所致蛙离体腹直肌收缩的影响。结果 NT能提高电刺激-嘶叫法,热板法的痛阈,抑制醋酸引起的扭体次数。NT镇痛具有剂量-效应关系,给药后2 h起效,5 h达峰。NT能使ACh引起的蛙腹直肌收缩的量效曲线平行右移。结论 NT对三种致痛模型均有确切的镇痛活性,NT对N2-AChR的拮抗作用为竞争性拮抗。     

眼镜蛇神经毒素的镇痛作用

眼镜蛇神经毒素(NT)在小鼠热板及大鼠电尾嘶叫测痛模型,都显示出明显的镇痛作用,并有剂量-效应关系。注射NT 0.022mg/kg,2h后,大鼠痛阈出现显著上升,3h后达到较佳效果,并能保持其效应强度达24h。连续给小鼠注射NT 0.023mg/kg,持续9d,小鼠并不产生耐受现象。结果提示,NT的镇痛机理可能与吗啡不同。     

眼镜蛇神经毒素大鼠不同脑区给药的镇痛作用研究

目的研究眼镜蛇神经毒素不同脑区给药的镇痛作用。方法大鼠30只分为对照组、中脑导水管组、第三脑室组、下丘脑组和尾状核组,对照组注射生理盐水组,第三脑室组注射10μL神经毒素,其他组注射0.5μL神经毒素,采用热水浴甩尾法测定痛阈。结果与对照组相比,其他组大鼠痛阈显著提高(P<0.01)。结论眼镜蛇神经毒素具有很强的中枢性镇痛作用,下丘脑部位注射效果最强。     

眼镜蛇神经毒素对豚鼠颈上神经节烟碱传递的易化作用(英文)

本研究用离休豚鼠颈上神经节细胞内生物电记录技术,对眼镜蛇毒神经毒素对交感神经节突触传递影响进行探讨。研究首次表明,眼镜蛇神经毒素对交感神经突触前ACh的释放有易化作用。     

眼镜蛇神经毒素粉末剂的鼻黏膜纤毛毒性考察

目的:考察浙江产眼镜蛇神经毒素鼻用粉末剂(神经毒素粉剂)的鼻黏膜纤毛毒性。方法:以在体蟾蜍上腭模型,观察神经毒素粉剂对鼻纤毛摆动的影响;运用组织病理学方法,在给于家兔神经毒素粉剂后1,3,5,7d时,取鼻中隔黏膜进行光学显微镜观察。结果:神经毒素粉剂抑制鼻纤毛摆动强度,但停止用药后8~9h纤毛摆动可完全恢复,且纤毛形态无明显改变;家兔给予神经毒素粉剂后1,3,5d,其鼻中隔黏膜与空白对照组差异无显著性。第7天,黏膜上皮松化,黏膜轻度充血,但基底膜完整。结论:神经毒素粉剂无急性不可逆性鼻黏膜纤毛毒性。     

眼镜蛇短链神经毒素镇痛和抗吗啡依赖作用实验研究

目的：眼镜蛇短链神经毒素（Cobratoxin，CTX）是从眼镜蛇蛇毒中提纯的短链α-神经毒素，是nACHR拮抗剂，特别是α7-受体亚型拮抗剂。本研究考察CTX是否有抗炎性疼痛和吗啡依赖的作用，并初步探讨其可能的作用机制。方法：采用5％福尔马林足皮下注射造成大鼠急性炎性疼痛和完全弗氏佐剂致大鼠慢性炎症模型，考察CTX是否具有抗炎性疼痛的作用；采用递增给药方式建立吗啡依赖小鼠和大鼠模型以及行为敏化模型探讨CTX的抑制吗啡依赖作用。     

转铁蛋白受体单克隆抗体OX26修饰的眼镜蛇神经毒素脂质体的制备及其对小鼠的镇痛作用研究

目的:制备转铁蛋白受体单克隆抗体OX26修饰的眼镜蛇神经毒素(αCT)脂质体(OX26-αCT-LP),考察其理化性质、体外释放行为,并研究其镇痛作用。方法:采用薄膜分散-挤出法制备αCT-LP,巯基化后的OX26再与脂质体相连制得OX26-αCT-LP。考察所制脂质体的形态、粒径、Zeta电位;采用透析袋法考察OX26-αCT-LP和αCT-LP在p H 7.4的磷酸盐缓冲液中60 h内的释放特性;采用小鼠热板法考察鼻黏膜滴注生理盐水、αCT原料药、αCT-LP和OX26-αCT-LP对小鼠舔后足反应潜伏期的影响。结果:所制OX26-αCT-LP形态呈球形,均匀圆整,平均粒径为(106.3±5.36)nm,Zeta电位为(-23.17±1.14)m V,药物包封率为(56.82±1.18)%;OX26-αCT-LP和αCT-LP的体外释药行为均符合Weibull方程(r=0.986 7、0.981 0);与生理盐水比较,αCT-LP和OX26-αCT-LP给药后60~480 min内能明显延长小鼠舔后足反应的潜伏期(P<0.01或P<0.05),且OX26-αCT-LP效果强于αCT-LP(P<0.01)。结论:成功制得对小鼠有一定镇痛作用的OX26-αCT-LP。     

浙江眼镜蛇(Naja naja atra)蛇毒镇痛多肽的分离纯化及其性质的研究

研究浙江眼镜蛇(Naja naja natra)蛇毒的镇痛活性组分，为寻找镇痛效果好，而无成瘾性的新型镇痛药奠定基础。采用CM-Sephadex离子交换色谱和Sephadex G-50凝胶过滤色谱对浙江产眼镜蛇蛇毒中的镇痛活性组分进行分离纯化。采用PAGE IEF及HPLC上等实验方法对产物纯度进行鉴定。采用SDS-PAGE法和HPLC法测定产物的分子质量，用IEF法测定产物的等电点，用小鼠热板法与醋酸扭体法考察产物的镇痛活性。结果表明眼镜蛇毒经3次柱色谱后，产物AAP经鉴定为单一组分。AAP经SDS-PAGE法和HPLC法测定的分子质量分别是7．28Mr和7．25Mr，等电点是9．58。AAP的痛阈提高百分率和扭体抑制率分别为91．3％，77．2％。眼镜蛇毒经3次柱色谱后得到具有镇痛活性的单一组分。     

Improved method for the isolation, characterization and examination of neuromuscular and toxic properties of selected polypeptide fractions from the crude venom of the Taiwan cobra Naja naja atra

An improved chromatographic method was developed to isolate and purify polypeptides and proteins from the crude venom of the Taiwan cobra Naja naja atra. The procedure devised is simple, easy to reproduce, and enables large scale isolation of almost all polypeptides and proteins in this cobra venom. Six pure polypeptide fractions of the venom were isolated and characterized using gel filtration on Sephadex G50 (medium), ion exchange chromatography on SP-Sephadex C25, desalting on Sephadex G25 (fine) and preparative HPLC on a RPC 18 column. The neuromuscular activity of these fractions was tested on the chick biventer cervicis nerve-muscle preparation and their toxicity (LD₅₀) was determined after i.v. administration in mice. Their antinociceptive activity was tested in the mouse abdominal test by i.v. application. Two of these polypeptide samples had major physiological effects: one acted as a cardiotoxin causing reversible myocardial contractures with no effect on muscle twitches elicited by nerve stimulation (NS); another was a neurotoxin that blocked muscle contractions in response to NS and exogenously added acetylcholine. The cardiotoxic fraction was identified as CTX I, a well-known cardiotoxin present in this venom, and the neurotoxin was identified as neurotoxin-α with an LD50 in mice of 0.075 mg/kg.     

Use of erythrocyte hemolysis kinetics in the purification of complex cardiotoxin mixtures

A human erythrocyte hemolysis kinetic method provides a useful way to follow the purification of cobra venom cardiotoxins or other hemolytic factors. Initial rates of hemolysis, measured as hemoglobin released with time for the separated cardiotoxins from the venom of the Thailand cobra Naja naja siamensis, vary over a greater range than do other commonly used measures of their biological activity. Recovery of the hemolytic activity of the gross cardiotoxin fraction in the subsequently separated fractions has been demonstrated. The method employs submilligram quantities of mixed or purified cardiotoxins.     

Biochemical characterization of a toxin from Indian cobra (Naja naja naja) venom

A major toxic component was isolated from the venom of Indian cobra (Naja naja naja) by ammonium sulfate fractional precipitation followed by carboxymethyl cellulose column chromatography and Sephadex gel filtration. This component constituted 2% of the venom and produced a block of neuromuscular transmission in nerve muscle preparations. Three other toxic fractions comprising 3% of the venom were also detected. The major toxic component was homogeneous on starch and polyacrylamide gel electrophoresis and on rechromatography on CM-cellulose. Its molecular weight was approximately 6300. This toxin contained 61 amino acid residues including 8 half-cystine residues whereas alanine, methionine and phenylalanine were totally absent. Its ld₅₀, as determined by i.p. injection in mice, was 0·2 mg per kg body weight. The fraction did not possess any enzymatic, hemolytic or hemagglutinin activities of crude venom but showed a close resemblance to the major neurotoxin of Formosan cobra venom.     

眼镜蛇蛇毒中神经毒蛋白对PC-12细胞作用的初步研究

目的:研究眼镜蛇蛇毒中的神经毒蛋白对大鼠肾上腺嗜铬细胞瘤细胞(PC-12细胞)的作用.方法:采用SP-SephadexC-25层析的方法分离出神经毒蛋白;MTT法检测神经毒蛋白对PC-12细胞的抑制率.结果:随着神经毒蛋白浓度的增加,对PC-12细胞的抑制率显著增加;随着时间的增长,对PC-12细胞的抑制率也明显增加....     

Investigation of the Anti-Snake Venom Activity of Schumanniophyton magnificum

Polar fractions of the methanolic extracts of the stem- and root-barks of SCHUMANNIOPHYTON MAGNIFICUM (Rubiaceae) reduced the effects of cobra venom cardiotoxin in the chick biventer cervicis nerve-muscle preparation IN VITRO. No activity was observed against a curaremimetic neurotoxin from cobra venom. Further fractionation of the extracts showed that the activity was not due to alkaloidal components, but the active substances could not be identified or isolated.     

Sequence characterization of venom toxins from Thailand cobra

Several toxins with distinct pharmacological properties were isolated from the venom of Thailand cobra (Naja naja siamensis) by cation-exchange chromatography. Two neurotoxins and one basic toxin with cardiotoxic activity were further purified and sequenced. The neurotoxins characterized were closely similar to the previously reported long- and short-chain neutrotoxins. The complete sequences of one minor neurotoxin and one cardiotoxin analogue were determined with the automatic protein sequencer in non-stop single runs of Edman degradation coupled with C-terminal sequence determination with carboxypeptidase digestion. The minor neurotoxin consists of 62 amino-acid residues with 8 cysteine residues and is found to be almost identical to cobrotoxin, a major toxic component of Formosa cobra (Naja naja atra). The sequence comparison of the 60-residue cardiotoxin with other reported cytotoxins of snake venoms indicates that 8 cysteine residues at the positions 3, 14, 21, 38, 42, 53, 54, and 59 are invariant among all sequences, with only two conservative changes at other positions along the sequence. The upshot of this report exemplified the facile sequence analysis of venom toxins by the application of pulsed-liquid phase protein sequencer and also revealed new analogues of a minor neurotoxin and one major cardiotoxin reported previously on the same species of Thailand cobra.     

金环蛇(Bungarus fasciatus)蛇毒的分离及其毒性组分的药理研究

金环蛇毒用羧甲基纤维素柱层析分离出17个蛋白组分,其中5个组分是毒性较大的致死成分。在这5个组分中,Ⅺ、Ⅻ、ⅩⅤ是心脏毒,ⅩⅢ、ⅩⅣ是神经毒。心脏毒组分Ⅺ、ⅫⅩⅤ使离体大鼠心脏挛缩,心跳停止于收缩期;使小鸡离体颈二腹肌挛缩,最后致痉挛性麻痹;对兔眼结合膜有局部刺激作用,使结合膜充血水肿。组分ⅩⅤ还具有直接溶血作用。神经毒组分ⅩⅢ阻断大鼠、小鸡、青蛙神经肌肉标本的突触传递,标本对乙酰胆碱的反应消失,对直接刺激以及对高浓度氯化钾的反应无减少,神经末梢乙酰胆碱的释放最无显著改变;使蛙腹直肌乙酰胆碱量效曲线平行右移,新斯的明可以对抗这个作用。组分ⅩⅣ的作用与组分ⅩⅢ相似。实验提示,组分ⅩⅢ、ⅩⅣ是作用于终板后膜的神经毒。     

Effectiveness of Thai cobra (Naja kaouthia) antivenom against sea snake (Lapemis hardwickii) venom: verification by affinity purified F(AB')2 fragments

Commercial Thai cobra (Naja kaouthia) antivenom was found to be effective in neutralizing sea snake (Lapemis hardwickii) venom. Neurotoxin specific F(ab')2 fragments obtained from the antivenom by chromatography using Thai cobra neurotoxin or sea snake venom affinity columns were able to neutralize both venoms.