乌梢蛇血清的抗出血因子:一个有前途的抗蛇毒药物原料

用柱层析和聚丙烯酰胺凝胶盘状电泳法,从乌梢蛇血清中分离纯化了一个抗出血因子。用SDS-聚丙烯酰胺凝胶电泳法测得其分子量大约为65 kD;测定了五种蝮亚科蛇毒(尖吻蝮、竹叶青蛇、原矛头蝮、哈扑和短尾蝮)的最小出血剂量和乌梢蛇血清中抗出血因子对这五种蛇毒的抗出血活性;还测定了七种蛇毒(除上述五种毒蛇外,还包括圆斑蝰和银环蛇)的半数致死量,以及抗出血因子对中毒小鼠的治疗作用。结果显示:从乌梢蛇血清中提纯的抗出血因子的抗蛇毒活性,不仅可以抵抗它的捕食者尖吻蝮的蛇毒,而且还可以抵抗具出血活性的其它蛇毒;但它对不具出血活性的银环蛇毒的致死抑制作用不明显。该抗出血因子不仅在体外实验表现出强的中和出血毒素的活性,而且在体内实验中亦表现出对中毒小鼠良好的治疗作用,因而可能成为新的抗蛇毒药物的有前途的原料。乌梢蛇血清对血循毒的中和能力的获得,可能归因于尖吻蝮与乌梢蛇之间捕食与被捕食相互作用的关系。     

原矛头蝮蛇毒对小鼠器官形态的影响

目的:观察原矛头蝮(P.mucrosquamatus)蛇毒对小鼠脏器形态结构的影响,从组织学角度探讨该蛇毒的致病机理。方法:用原矛头蝮攻击小鼠,待小鼠死亡后,立即进行解剖,观察心脏和大血管内部血栓形成情况。取心脏、血管、周围神经、肾脏、肝脏、脾脏和肺,观察外形,并进行石蜡切片,H.E染色观察。用健康小鼠相同器官为对照,比较二者的差异。结果:被原矛头蝮攻击致死小鼠的心脏、肝脏和肺发生了显著收缩;心腔和血管腔被血栓堵塞;在心脏、肝脏和肾脏组织血管中观察到了血栓;脾脏中白细胞显著增多;肺泡收缩,细支气管严重收缩致管腔堵塞;股动脉血管内膜发生了脱落;在股神经中发生了脱髓鞘现象。结论:急性凝血和窒息是导致小鼠死亡的重要原因;原矛头蝮蛇毒兼具血循毒和神经毒的特征;原矛头蝮蛇毒可导致多种脏器内部结构发生改变。     

尖吻蝮咬伤中毒的机制与治疗

尖吻蝮Dienagkistrodon acutus(Guenther)俗称五步蛇,是危害劳动人民较严重的一种毒蛇。分布于安徽、浙江、江西、福建、台湾、湖北、湖南、广东、广西、贵州。国外分布于越南北部。被咬后,若治疗不及时或抢救不力,往往使病情转重,造成肢体残废,甚至危及生命。蛇毒对机体的毒理作用尖吻蝮隶属于蝮亚科,是有颊窝(感觉温度的器官)的管牙类毒蛇。根据我国广西报告,平均每条蛇咬物一次排出的毒液222.2毫克(相当于59.0毫克干毒量),对小白鼠的半数致死量8.9毫克/公斤,皮下注射,其毒性虽较弱,但其排毒量甚多,对这种蛇伤的危险性也就不可勿视。尖吻…     

基于蛇毒提取物的超级止血粘合剂的发现

正蛇毒可以伤人,也可以救人。它的主要毒性(活性)成分是蛋白质,根据作用部位及原理不同一般分为神经毒素、心脏毒素、凝血毒素、出血毒素等。研究人员分离和研究了多种毒蛇的毒液,目前基于蛇毒毒素也开发出了多种抗癌、镇痛、抗血栓的药物。受蛇毒凝血功能的启发,来自陆军军医大学西南医院联合加拿大曼尼托巴大学的研究团队将从巴西矛头蝮Bothrops atrox的毒液中提取的血凝酶(hemocoagulase,HC)引入明胶-甲基丙烯酸凝胶(Gel MA),     

乙醇对中华眼镜蛇毒毒性的影响

目的为了探索乙醇对眼镜蛇毒毒性的影响。方法将眼镜蛇毒不同浓度致死量经不同浓度乙醇体外处理后,分别于小白鼠皮下注射、口服,将致死量蛇毒皮下注射后的小白鼠立即于局部注射乙醇,观察蛇毒毒性情况。结果小白鼠经皮下注射致死量眼镜蛇毒后,在局部注射50％（或异蛇米酒）、75％乙醇0．1～0．2ml有一定的保护作用;口服100倍皮下注射致死量眼镜蛇毒未发现有毒性表现,口服经50％乙醇处理后的眼镜蛇毒（100倍皮下注射致死量）未增加小鼠死亡率。结论眼镜蛇毒体外经过乙醇处理后毒性有所下降。口服少量的眼镜蛇毒是安全的。眼镜蛇毒与乙醇混合后口服未见蛇毒毒性增加。     

蛇毒丝氨酸蛋白酶多样性──底物专一性免疫化学及序列比较研究

本文报道烙铁头（Ｔｒｉｍｅｒｅｓｕｒｕｓｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒纤维蛋白原溶酶（ＴＭＶＦｇ），眼镜王蛇（Ｏｐｈｉｏｐｈａｇｕｓｈａｎｎａｈ）蛇毒纤维蛋白原溶酶（ｏｈＳ１），竹叶青（Ｔｒｉｍｅｒｅｓｕｒｕｓｓｔｅｊｎｅｇｅｒｉ）蛇毒专一纤溶酶原激活剂（ｓｖ－ｐＡ）对５种小分子多肽底物的底物专一性，及这些蛇毒丝氨酸蛋白酶对各种凝血因子（第Ｘ因子、凝血酶原、纤溶酶原、蛋白Ｃ）的作用，并和其它蛇毒丝氨酸蛋白酶如矛头蝮（Ｂｏｔｈｒｏｐｓａｔｒｏｘ）蛇毒凝血酶样酶（Ｂａｔｒｏｘｏｂｉｎ）、铜头蝮（Ａｇｋｉｓｔｒｏｄｏｎｃｏｎｔｏｒｔｒｉｘｃｏｎｔｏｒｔｒｉｘ）蛇毒蛋白Ｃ激活剂ＡＣＣ－Ｃ、蝰蛇（Ｖｉｐｅｒａｒｕｓｓｅｌｌｉ）毒第Ⅴ因子激活剂ＲＶＶ－Ｖ进行比较研究。通过酶标偶联免疫反应研究了抗ｓｖ－ＰＡ抗体与各种丝氨酸蛋白酶的免疫交叉反应，并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。     

一次接种HA DNA保护小鼠抵抗B型流感病毒攻击

为详细探讨小鼠不同次数接种B型流感病毒HA DNA疫苗后免疫应答情况,以及CpG基序的免疫佐剂效果,采用不同剂量HA DNA,1次或2次(间隔3周)电击法免疫BALB/C小鼠。初免4周后(或二免加强免疫1周后)用致死量流感病毒(B/Ibaraki/2/85)攻击。研究发现:①100μg HA DNA一次接种的小鼠全部存活;②经含有CpG基序的DNA免疫的小鼠体内诱导产生的抗HAIgG抗体更高,小鼠体重丢失更少。这些结果说明,1次接种100μg HA DNA疫苗可以使小鼠抵抗致死量B型流感病毒攻击,CpG基序能够增强HA DNA疫苗的免疫保护作用。     

尖吻蝮蛇毒研究进展

蛇毒是从毒蛇头部毒腺分泌的有毒液体,是一种重要的天然动物毒素,其主要成分是蛋白质,具有广泛的生物学活性,现已被应用于溶栓、抗癌、止血、止痛等方面。传统上蛇毒分为神经毒、血循毒与混合毒。尖吻蝮是我国十大剧毒蛇之一,其毒液主要作用于血液系统,引起出血、肿胀与局部组织坏死。已经发现尖吻蝮蛇毒多属丝氨酸蛋白酶、金属蛋白酶和磷脂酶屯家族,部分蛋白质可能属于去整合素家族[1]。现就尖吻蝮蛇毒生理活性及药理应用等方面的研究进展综述如下。     

蛇毒合成的信号通路

蛇毒液产生的周期分为活跃期和静息期两个阶段,在这两个阶段中毒腺分泌细胞的形态学和生物化学方面存在着许多不同。蛇咬物排毒或人工取毒后其释放的去甲肾上腺素(noradrenaline,NE)是分泌细胞中合成毒液所必需的,其中分泌细胞的α肾上腺素能受体(α-adrenoceptor,α-AR)和β肾上腺素能受体(β-adrenoceptor,β-AR)参与了这个过程。本文简要介绍在毒液产生周期的不同阶段中分泌细胞的变化,重点阐述了刺激α-AR和β-AR在引起毒液合成开始时所介导的相关细胞信号通路。最后简单探讨了其它刺激蛇毒腺引起毒液产生的可能因素及其调控机制。     

四种不同方法注射眼镜蛇蛇毒对小鼠LD_(50)的影响分析

目的比较小鼠经不同途径注射眼镜蛇蛇毒的半数致死量(LD_(50)),并观察中毒症状。方法将小鼠分别经四种途径注射眼镜蛇毒冻干粉溶液,并根据各组小鼠死亡情况,运用Bliss软件测定LD_(50)估计值、置信区间等。观察各组小鼠生物学行为及死亡情况,并对死亡小鼠和存活小鼠进行解剖分析。结果小鼠经静脉、腹腔、肌肉和皮下注射眼镜蛇蛇毒的LD_(50)分别为0.691 mg/kg、0.741 mg/kg、0.803 mg/kg和0.915 mg/kg,95%CI分别为0.634～0.742 mg/kg、0.676～0.792 mg/kg、0.754～0.856 mg/kg和0.851～1.05 mg/kg。各组小鼠中毒症状明显,表现为呼吸困难、精神萎靡、眼球昏暗、眼角有分泌物、身体出现扭体抽搐等反应,均在2～9 h内死亡。结论小鼠经静脉注射眼镜蛇毒溶液中毒的毒性剂量最小,皮下注射眼镜蛇毒溶液中毒的毒性剂量最大。小鼠经四种不同途径注射眼镜蛇蛇毒的LD_(50)的值按从大到小的排序为皮下注射,肌肉注射,腹腔注射,静脉注射。     

Snake venomics and antivenomics of Protobothrops mucrosquamatus and Viridovipera stejnegeri from Taiwan: Keys to understand the variable immune response in horses

The proteomes of the venoms of the snakes Viridovipera stejnegeri and Protobothrops mucrosquamatus from Taiwan were characterized by N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of in-gel generated tryptic peptides. Proteins belonging to the following toxin classes were identified: metalloproteinase, phospholipase A(2) (PLA(2)), serine proteinase, C-type lectin-like, CRISP, l-amino acid oxidase, disintegrin, and peptides (vasoactive and inhibitors of SVMPs). Nine horses were immunized with a mixture of these venoms. All horses developed a satisfactory immune response against lethality of the venom of V. stejnegeri, whereas only three horses reached the accepted neutralizing potency against the venom of P. mucrosquamatus. Antivenoms were prepared from pools of 'good responder' (GR) and 'poor responder' (PR) horses and compared by antivenomics and neutralization tests. A similar neutralizing response was observed between the GR and PR antivenoms against the venom of V. stejnegeri, whereas antivenom from PR had a lower neutralizing activity against effects of P. mucrosquamatus venom than antivenom from GR. The low potency of the plasma of some horses against this venom is a consequence of the low immunogenicity of the neurotoxic PLA(2) trimucrotoxin. Our results provide clues for innovating the immunization scheme to generate improved antivenoms.     

急危重症眼镜蛇伤大鼠多项凝血指标动态变化

目的动态观察蛇伤后大鼠多项凝血指标、病理及皮下出血情况的改变,为蛇毒毒理及抗蛇毒制剂的研究提供实验数据。方法将SD大鼠注入2～4 LD50眼镜蛇毒造模,于注毒后不同时段(20～140 min)分别进行凝血功能、肝脏和肾脏病理等检测,处死大鼠后观察注毒部位皮下出血情况。结果大鼠注入4 LD50蛇毒20 min,凝血酶原时间(PT)、部分凝血酶原时间(APTT)延长;40 min时,纤维蛋白原(Fbg)明显下降;40 min后凝血功能逐渐回复至0 min水平。肾脏、肝脏组织见血管扩张、充血明显。注毒部位皮下出血范围于20～80min时最明显。2 LD50剂量组凝血功能的变化与4 LD50组相似,但其Fbg下降程度小于4 LD50组,皮下出血面积较4 LD50组小。结论眼镜蛇毒引起凝血功能改变,且其变化程度与剂量及注入时间密切相关。     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup.     

The variability of Vipera ammodytes ammodytes venoms from Croatia--biochemical properties and biological activity

Vipera ammodytes ammodytes venom has been used for many years in Croatia for immunization of horses and production of specific therapeutic anti-venoms. The neutralizing effectiveness of anti-venoms is directly dependent on the properties of the snake venom used for immunization. Therefore, appropriate characterization of the whole venom is necessary prior to use in the immunization procedure. In the course of such analyses, the variability in biochemical properties and biological activity was observed in venoms collected from snakes originating from different parts of Croatia. The venom pools also differed with respect to time of snake collection (1992-2003). Analyses of three samples of whole venom pools were carried out revealing differences in lethal activity (LD50), minimum haemorrhagic dose (MHD), minimum necrotizing dose (MND), phospholipase A2 activity and in anticomplementary activity. SDS-PAGE electrophoretic patterns were similar, but not identical, for all tested venom pools with respect to the number of protein bands detected, but intensity of particular components differed. Preliminary immunogenicity testing in terms of determination of specific antibodies revealed similar immunogenicity and high cross-reactivity for three samples tested.     

Feeding behavior and venom toxicity of coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity

The feeding behavior and venom toxicity of the coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity were investigated. Coral snakes searched for their prey (the colubrid snake Geophis godmani) in the cages. Once their preys were located, coral snakes stroke them with a rapid forward movement, biting predominantly in the anterior region of the body. In order to assess the role of venom in prey restraint and ingestion, a group of coral snakes was 'milked' in order to drastically reduce the venom content in their glands. Significant differences were observed between snakes with venom, i.e., 'nonmilked' snakes, and 'milked' snakes regarding their behavior after the bite. The former remained hold to the prey until paralysis was achieved, whereas the latter, in the absence of paralysis, moved their head towards the head of the prey and bit the skull to achieve prey immobilization by mechanical means. There were no significant differences in the time of ingestion between these two groups of coral snakes. Susceptibility to the lethal effect of coral snake venom greatly differed in four colubrid species; G. godmani showed the highest susceptibility, followed by Geophis brachycephalus, whereas Ninia psephota and Ninia maculata were highly resistant to this venom. In addition, the blood serum of N. maculata, but not that of G. brachycephalus, prolonged the time of death of mice injected with 2 LD(50)s of M. nigrocinctus venom, when venom and blood serum were incubated before testing. Subcutaneous injection of coral snake venom in G. godmani induced neurotoxicity and myotoxicity, without causing hemorrhage and without affecting heart and lungs. It is concluded that (a) M. nigrocinctus venom plays a role in prey immobilization, (b) venom induces neurotoxic and myotoxic effects in colubrid snakes which comprise part of their natural prey, and (c) some colubrid snakes of the genus Ninia present a conspicuous resistance to the toxic action of M. nigrocinctus venom.     

Studies on a murine model for evaluation of virulence of Streptococcus suis capsular type 2 isolates

Five different parameters, time of incubation of the culture, type of culture medium, inoculum, strain of inbred mice, and age of mice, were tested using the LD50 technique to standardize a murine model for the evaluation of the virulence of Streptococcus suis capsular type 2 isolates. A model using 28 day-old mice belonging to CF1 strain appeared to give the best results. The inoculum size was the parameter most influencing the 50% lethal dose obtained with mice. Inoculation with 1-ml volume of a bacterial suspension instead of 0.1 or 0.5 ml decreased the LD50. The standardized model was used to evaluate the virulence of some isolates of known pathogenicity for pigs. The minimum lethal dose was used in the model and it appeared that the virulence of Streptococcus suis capsular type 2 isolates can be measured from highly virulent to totally avirulent.     

乙醇对五步蛇（Agkistrodonacutus）毒毒性的影响

目的为了探索乙醇对五步蛇毒毒性的影响。方法将五步蛇毒不同浓度致死量经不同浓度乙醇体外处理后,选择小白鼠分别给予皮下注射、口服,并将致死量蛇毒于小白鼠皮下注射后立即于局部注射乙醇,观察蛇毒毒性情况。结果使用乙醇体外处理后的五步蛇毒毒性明显下降,小白鼠经皮下注射致死量五步蛇毒后立即在局部注射50％异蛇米酒或75％乙醇0．1～0．2ml有一定的保护作用,口服20倍致死量五步蛇毒未发现有毒性表现。结论五步蛇毒体外经过乙醇处理后毒性明显下降。口服五步蛇毒是安全的。蛇毒进入皮下后立即用乙醇处理有一定的治疗效果。     

蛇毒酶制剂出血毒试验方法比较

使用不同稀释度的蛇毒毒素,等体积注射于大鼠背部皮下、皮内和小鼠背部皮下,１８～２４小时剥皮观察比较动物皮下出血程度,经统计学处理,小鼠背部皮下注射对检查出血毒最敏感。选用小鼠背部皮下注射０．２ｕ蛇毒酶成品、半成品,１８～２４小时剥皮观察皮下瘀血或瘀斑,发现４２批成品中８８．１％未见皮下瘀血,２４批半成品７５％未见皮下瘀血。说明选用小鼠背部皮下注射０．２ｕ蛇毒酶来限量检查出血毒的方法是可取的。