眼镜蛇毒组分C的抗瘤活性及其对肿瘤细胞存活率的影响

目的观察眼镜蛇毒组分Ｃ对ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２的抑瘤作用及其体外对肝癌Ｈ２２细胞存活率的直接影响。方法通过半体内实验,观察眼镜蛇毒组分Ｃ对小鼠腹水型肝癌Ｈ２２的抑瘤率;通过细胞培养,以细胞存活率为指标观察中华眼镜蛇毒组分Ｃ对ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２细胞在体外的直接杀伤作用。结果眼镜蛇毒组分Ｃ能明显抑制ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２细胞的生长,其抑瘤作用随剂量增大而增强,ＩＣ５０为９５ｍｇ／Ｌ。在体外,眼镜蛇毒组分Ｃ的浓度对ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２细胞存活率的影响随其作用剂量的增大而增强,ＩＣ５０为９ｍｇ／Ｌ;同时,这种影响还随其作用时间的延长而增强。结论眼镜蛇毒组分Ｃ对ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２有显著的抑瘤作用。     

眼镜蛇毒及其组分C对大鼠实验性肝癌抗癌作用的病理学研究

目的 观察眼镜蛇毒及其组分Ｃ抗小鼠肝癌的病理学改变。方法 采用不同剂量眼镜蛇毒及其抗癌活性组分Ｃ与ＢＡＬＡ／ｃ小鼠腹水型肝癌细胞体外孵育,空白对照组用生理盐水与肝癌细胞孵育,然后取孵育液接种于小鼠前肢腋下,接种后第１０ｄ坏死,解剖取出瘤结,进行病理组织学研究。     

眼镜蛇毒及其组分C对小鼠实验性肝癌抗癌作用的病理学研究

目的　观察眼镜蛇毒及其组分 Ｃ 抗小鼠肝癌的病理学改变。 方法　采用不同剂量眼镜蛇毒及其抗癌活性组分 Ｃ 与 Ｂ Ａ Ｌ Ａ／ｃ 小鼠腹水型肝癌细胞体外孵育, 空白对照组用生理盐水与肝癌细胞孵育, 然后取孵育液接种于小鼠前肢腋下, 接种后第 １０ｄ 处死, 解剖取出瘤结, 进行病理组织学研究。 结果　空白对照组瘤结较大, 显微镜下见瘤细胞生长活跃、核大、核仁明显、核分裂多见, 而坏死灶少见, 且瘤细胞向周围浸润扩散; 治疗组瘤体较小, 瘤细胞固缩、核仁不明显、核分裂少见, 而坏死灶多见, 瘤细胞周围有纤维组织增生围绕, 限制了瘤细胞向周围蔓延浸润。 结论　眼镜蛇毒及其组分 Ｃ 对小鼠实验性肝癌的体外抗癌作用是明显的, 不同剂量及不同孵育时间的抗癌作用亦显著不同, 其中组分 Ｃ 对抗癌作用最强。     

南蛇簕抗眼镜蛇毒的实验研究

目的探讨南蛇簕抗眼镜蛇毒的作用。方法抗眼镜蛇毒采用鲎试剂试验法。结果眼镜蛇毒与0.5EU/ml鲎试剂产生凝集反应的浓度在7.81μg/ml以上;南蛇簕提取液(含生药)为0.5g/ml浓度时可抗10倍量眼镜蛇毒的凝集反应。结论草药南蛇簕有较强的抗眼镜蛇毒作用。     

蛇伤药酒抗毒抗菌作用的实验研究

目的探讨蛇伤药酒抗眼镜蛇毒、内毒素及细菌的效果。方法抗眼镜蛇毒和内毒素试验采用鲎试剂法．抗菌试验采用纸片法。结果眼镜蛇毒与0．5EU／ml鲎试剂产生凝集反应的浓度在5．0ug／ml以上;蛇伤药酒浓度1ml／ml时有抗5倍量眼镜蛇毒的凝集反应;有抗2．5倍量内毒素的凝集反应;对多种细菌有抑制作用。结论蛇伤药酒有抗眼镜蛇毒、抗内毒素和多种病原菌的作用。     

眼镜蛇毒研究与临床应用近况

用眼镜蛇整体泡酒饮服,治疗腰腿痛,祖国医学已有记载［１］,已有悠久历史。３０年代国外应用眼镜蛇毒治疗关节痛、神经痛,临床应用数百例,后因起效慢等原因,而终止研究与应用。我院于６０年代初开始对眼镜蛇毒进行系统研究,如原料标准,注射剂质控标准、安全试验、半数致死量（ＬＤ５０）试验、药理学、药效学、长毒以及“三致”试验等。结果表明：蛇毒及其制剂,治疗风湿痛、神经痛等疗效高于毒副反应。同时我们又研究以眼镜蛇毒为君药与中药伍用,治疗肝癌及消化道癌,亦收到了较好的疗效。这对深入研究与开发蛇毒新药是大有前途的     

黑皮蛇、山白菜抗五种蛇毒的实验研究

目的研究黑皮蛇、山白菜抗五种蛇毒的药理作用。方法采用鲎试剂凝胶法对黑皮蛇、山白菜抗五种蛇毒的作用进行实验研究。结果黑皮蛇药液浓度在0.4g/ml以上、山白菜药液浓度在0.2g/ml以上,均能抗五种蛇毒与鲎试剂的凝集反应。结论黑皮蛇、山白菜有较强的抗眼镜蛇毒、竹叶青蛇毒、蝰蛇毒、蝮蛇毒和五步蛇毒作用。     

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。     

抗中华眼镜蛇毒鸡卵黄抗体的制备及其效价测定

目的探索免疫鸡制备高效价抗眼镜蛇毒抗体的新方法。方法用中华眼镜蛇原毒作抗原免疫22周龄的莱航母鸡,水溶法粗提抗体,DEAE Sepharos FF柱纯化,切向流超滤膜脱盐及浓缩,免疫电泳及双向免疫扩散法进行鉴定及效价测定,采用BCATMProte in Assay K it测定蛋白含量。结果鸡卵黄经水溶法的粗提物与中华眼镜蛇毒即有较明显沉淀反应,其效价随着纯度的提高而增强。将马源性抗血清的蛋白质含量调至与浓缩的IgY相同(2mg/m l),经双向免疫扩散及免疫电泳鉴定,该抗体不但对中华眼镜蛇毒有特异性结合,与孟加拉眼镜蛇毒亦有较强的交叉免疫活性,其效价较马抗眼镜蛇毒血清高4倍以上。结论用中华眼镜蛇原毒制备的IgY抗体,其效价较马抗血清有显著提高,并与孟加拉眼镜蛇毒有高度交叉免疫。本实验为抗眼镜蛇IgY的应用及其它抗蛇毒IgY的制备奠定了基础。     

青龙蛇药抗蛇毒中毒的实验研究

本文总结对青龙蛇药抗蛇毒中毒作用进行研究的买验结果,经动物实验证明,青龙蛇药口服对眼镜蛇毒、眼镜王蛇毒、五毒蛇毒及蝮蛇毒中毒的小白鼠有明显的保护作用,而对银环蛇毒中毒的保护率很低;还证明该药有对抗蛇毒的出血毒性、溶血毒性、毛细血管损伤及组织坏死的作用。     

眼镜蛇毒神经生长因子抑制肝星状细胞增殖并诱导其凋亡

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、凋亡活性的影响,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;6.25μg/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%(5μg/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%(5μg/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。     

Primary structure of cobra complement component C3.

Complement component C3 is a multifunctional protein known to interact specifically with more than 10 different plasma proteins or cell surface receptors. Cobra venom contains cobra venom factor, a structural analogue of C3 that shares some properties with C3 (e.g., formation of a C3/C5 convertase) but differs in others (e.g., susceptibility to regulation by factors H and I). The elucidation of structural differences between C3 and cobra venom factor can be expected to help identify functionally important regions of C3 molecules. To that end we have undertaken the molecular cloning of both cobra C3 and cobra venom factor to take advantage of the unique biologic system where both proteins are produced by the same species. We report the primary structure of cobra C3 mRNA and the derived protein structure. Cobra C3 mRNA is 5211 bp in length. It contains an open reading frame of 4953 bp coding for a single pre-pro-C3 molecule, consisting of a 22-amino acid signal sequence, a 633-amino acid beta-chain (70 kDa), and a 992-amino acid alpha-chain (112 kDa) which is separated from the beta-chain by four arginine residues. There are no N-glycosylation sites in cobra C3. Cobra C3 exhibits approximately 58% nucleotide sequence identity with C3 from mammalian species. At the protein level, sequence identity is approximately 52% and sequence similarity approximately 71%. All 27 cysteine residues are highly conserved as are the C3 convertase cleavage site, the thioester site, and the factor B binding site. Cobra C3 also seems to have homologous binding sites for factor H and properdin, as well as a conserved sequence in the functionally important region of the C3a anaphylatoxin. The sequence homology at the CR2 and CR3 binding sites does not exceed the overall sequence homology. Accordingly, the existence of CR2 and CR3 binding sites can neither be deduced nor excluded.     

Immunopharmacological Approach to Forssman Shock

Forssman shock (FS) following the intravenous injection of antisheep erythrocyte antibody into guinea pigs resulted in fatal systemic shock with marked decrease in CH₅₀ values of complement, leucocyte, and platelet counts, and a prolongation of blood coagulation time. In addition, there was an increase in lactate dehydrogenase activity, and decreases in both esterase activity and fibrinogen levels were noted. F(ab′)₂ of antisheep erythrocyte IgG antibody was not capable of eliciting FS. Cobra venom factor showed a fairly potent inhibition of FS. Leukopenia induced by cytosine arabinoside given intraperitoneally for 5 days had no effect on FS. Colchicine, which decreased the leucocyte count, did not inhibit fatal systemic shock. Administration of heparin or trasyrol did not prevent FS. The present findings demonstrate that FS is inhibited by anticomplementary agents but not by drugs which affect leucocyte and platelet counts, the coagulation system or serum proteases.     

Production of potent antivenin against cobra venom with conjugated-cobrotoxin.

Cobra venom (Naja naja atra) and its fractions obtained by ammonium sulfate precipitation were subjected to chromatography on CM-Cellulose colum. A highly purified cobrotoxin obtained by the repeated chromatography on preparative CM-Cellulose column was 6.7 times more toxic than the original cobra venom. The toxin was detoxified by a bifunctional reagent, glutaraldehyde, to about 99.8% and utilized for immunization in animals. Mice received 4 weekly immunization with detoxified cobrotoxin and challenged one week after the last injection showed 60% protection in rabbits by immunization with detoxified cobrotoxin reached 360 LD50 neutralizing level against the cobra venom within 30 days. The results indicate that it is feasible and promising to prepare potent antivenin in animals by glutaraldehyde-treated cobrotoxin.     

中华眼镜蛇蛇毒因子(CVF)对补体系统的作用及与人补作C_3和其他蛇毒抗原性关系

中华眼镜蛇蛇毒经DEAE-Sepharose CL-6B。HPLC等多次柱层析分离出有抗补体及溶血活性的眼镜蛇蛇毒因子(Cobra venom factor,CVF),纯化后的CVF在聚丙烯酰胺凝胶电泳图谱上呈单一区带,分子量为225000—230000,等电点为6.20。用二硫苏糖醇还原经SDS-聚丙烯酰胺凝胶电泳得三类亚基,其分子量总和为237,000。 体外抗补体及溶血试验表明,CVF的作用是通过补体旁路途经使总补体活力下降。双向免疫电泳鉴定,发现CVF与人血清作用后,其中补体成分C_3分子的抗原性发生改变,则表明CVF的作用是通过激活补体成分C_3而发挥的。给豚鼠腹腔注射CVF(0.15ug/g体重)后,其血清总补体水平下降到正常值的3％以下,7天后回升,13天后恢复到正常水平。 单相免疫电泳表明,CVF与人补体C_3抗血清间无任何交叉免疫反应,但人血清与CVF抗血清间有微弱的免疫沉淀反应。另外,CVF的氨基酸组成与人补体C_3也较为相似。鉴定还表明眼镜蛇科中四种蛇毒与CVF抗血清有强烈的免疫沉淀反应,蝰蛇毒及海蛇毒也有免疫沉淀反应,但只有眼镜蛇毒具有抗补体活性。     

八种常见国产蛇毒对白血病细胞杀伤作用研究

用体外细胞培养的方法,观察了我国常见的八种蛇毒（眼镜蛇毒、蝮蛇毒、眼镜王蛇毒、竹叶青蛇毒、蝰蛇毒、烙铁头蛇毒、银环蛇毒及金环蛇毒）对人白血病Ｔ淋巴细胞系ＣＥＭ细胞、人单核细胞白血病Ｕ９３７细胞及人早幼粒细胞白血病ＨＬ６Ｏ细胞的生长曲线、存活率及分裂指数的影响。结果发现八种常见蛇毒中眼镜蛇毒对白血病细胞的杀伤作用最强,蝮蛇毒次之（与空白对照组相比,Ｐ值均小于０．０１）,其余六种蛇毒的作用则很弱。但无论是眼镜蛇毒还是蝮蛇毒,其杀伤淋巴细胞白血病、单核细胞白血病及早幼粒细胞白血病细胞的作用之间无显著差异。     

Occurrence of non-protein low molecular weight cardiotoxin in Indian King Cobra (Ophiophagus hannah) Cantor 1836, venom

Pathophysiology due to snakebite is a combined effect of various actions of the complex venom constituents. Importance of protein toxins in snake envenomation is well known. The present investigation reports the existence of nonprotein/nonpetide low molecular weight toxin in Indian King Cobra venom, which plays an important role in envenomation consequences in experimental animal models. A group of non-peptidic toxins (OH-NPT1) was isolated from Indian King Cobra Ophiophagus hannah by thin layer chromatography and silica gel column chromatography. UV, IR, NMR and (ESI) TOF-MS studies characterized the OH-NPT1 as a mixture of aliphatic acids having molecular weights 256, 326 and 340Da. The minimum lethal dose of OH-NPT1 was found to be 2.5 microg/20g (iv) and 4microg/20g (ip) in male albino mice. The cardiotoxic property of OH-NPT1 was established through studies on isolated guinea pig heart and auricle preparations, ECG studies in albino rat and estimation of LDH1/LDH and CPK-MB/CPK ratio in Swiss albino mice. Commercial antiserum failed to neutralize the lethality and cardiotoxicity of the toxin. However, calcium and magnesium effectively neutralized the lethal action.     

Preparation and characterization of immunoglobulin yolk against the venom of Naja naja atra

Chinese Cobra (Naja naja atra) bite is one of the leading causes of snake-bite mortality in China. The traditional anti-cobra venom serum therapy was found to be expensive and with high frequency of side effects. Therefore attempts were made to generate a high titer immunoglobulin from egg yolk (IgY) of crude cobra-venom immunized Leghorn hens, and to standardize an effective method for producing avian antivenom in relatively pure form. The IgY was isolated first by water dilution method to remove the lipid, then extracted by ammonium sulfate precipitation, and purified through anion exchange chromatogram. The different purities of IgY from different isolating stages were submitted to enzyme-linked immunosorbent assay and SDS-PAGE to determine their titers. Immunoblotting showed that the purified IgY (ion exchange chromatography fraction, IECF) recognized several antigenic fractions of cobra venom, and presented with the character of polyclonal antibody. IECF on SDS-PAGE under reducing conditions migrated as a 65 kDa heavy chain and a 35 kDa light chain, respectively. The LD50 of the N. naja atra venom was 0.62 mg/kg body weight in mice. Four times the LD50 dose of venom was selected as challenge dose, and the ED50 of IgY was 3.04 mg IECF/mg venom. The results indicate that the activity of anti-snake venom IgY could be obviously elevated by ion exchange chromatography, thus possessing therapeutic significance for snakebite envenomation.     

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。     

Stimulation of purified phosphatidylinositol 4-kinase by cobra venom cardiotoxin

Cobra venom cardiotoxin was found to stimulate the phosphatidylinositol kinase activity present in A431 cell membranes and in detergent extracts of these membranes. Incubation of highly purified phosphatidylinositol 4-kinase from this source with cardiotoxin resulted in a 2- to 3-fold stimulation of phosphatidylinositol kinase activity. The activation of the purified phosphatidylinositol 4-kinase by cardiotoxin was time- and dose-dependent and appeared to be associated with a decrease in the Km apparent of the enzyme for phosphatidylinositol with no change in the Vmax apparent of the enzyme. The data suggest that the phosphatidylinositol 4-kinase is activated by direct interaction of the enzyme with cobra venom cardiotoxin.