舟山眼镜蛇毒细胞毒素的分离纯化及其体外抗肿瘤活性

目的从眼镜蛇毒中分离纯化细胞毒素 F(CTX F)并鉴定其活性。方法应用凝胶过滤、离子交换柱色谱及疏水柱色谱等方法从舟山眼镜蛇毒中分离纯化CTX F ,以SRB法观察CTX F对体外培养癌细胞的杀伤作用。结果眼镜蛇毒粗毒经凝胶过滤获得 4个蛋白峰 ,将CTX所在第Ⅳ峰用阳离子交换柱色谱获A、B、C、D、E、F和G等7个组分 ,其中E、F和G具CTX活性 ,将F组分再经凝胶过滤和疏水色谱进一步纯化得不含磷酯酶A2 (PLA2 )的CTX纯品 ,暂定名为CTX F ,它对多种癌细胞株有杀伤作用。结论应用凝胶过滤、离子交换和疏水色谱等方法可从眼镜蛇毒中获得不含PLA2 的CTX ,其组分F有抗肿瘤活性     

眼镜蛇毒细胞毒素的分离、纯化及其抗癌活性

目的：研究眼镜蛇毒细胞毒素的抗癌活性。方法：应用Ｓｅｐｈａｄｅｘ　 Ｇ１００和 ＣＭ－Ｓｅｐｈａｒｏｓｅ ＦＦ柱层析,从眼镜蛇毒中分离、纯化细胞毒素组分,用ＭＴＴ法测定该组分对体外培养的人癌细胞的细胞毒性作用。结果：眼镜蛇毒细胞毒素对人癌细胞ＳＧＣ－７９０１、Ｂｅｌ－７４０２、Ｋ５６２和Ｕ９３７的抑制作用呈良好的量效关系,半数抑制浓度分别为 ４．１０、 ２． ０８、 ０． ２９和 ０．１７ μｇ／ｍｌ。结论：眼镜蛇毒细胞毒素对体外培养的人癌细胞有很强的杀伤作用。     

眼镜蛇毒细胞毒素缓释微球制备及体外性质研究

目的研究眼镜蛇毒细胞毒素(Cytotoxin,CTX)聚乳酸/羟基乙酸缓释微球的制备方法,考察其一般性质、体外释药特性及生物学活性。方法采用色谱方法纯化眼镜蛇CTX,MTT方法检测细胞毒活性,复乳-溶剂挥发法制备载药微球,考察微球表面形态、粒径、包封率、载药率、体外释药行为及释放眼镜蛇CTX细胞毒活性。结果纯化眼镜蛇CTX具有明显的细胞毒作用,对肝癌HepG2细胞12,24 h的IC50分别为1.43,1.12μg/mL,对L02肝细胞12,24 h的IC50分别为1.37,1.01μg/mL。微球表面光滑圆整,粒径2.1~7.8μm,包封率和载药率分别为(74.10±9.92)%和(0.72±0.09)%,30 d药物累积释放84.3%,释放眼镜蛇CTX保持较好的生物学活性。结论采用复乳-溶剂挥发法可制备具有较高包封率,良好缓释效果,保持完整生物学活性的眼镜蛇CTX缓释微球。     

眼镜蛇毒L-氨基酸氧化酶分离纯化及对血管内皮细胞的抑制作用

目的从眼镜蛇毒中分离纯化L-氨基酸氧化酶(L-a-mino acid oxidase of Naja atra venom,NAV-LAAO),观察其对血管内皮细胞是否有抑制作用。方法应用阳离子交换层析和亲和层析方法分离纯化NAV-LAAO;采用MTT法、Western blot测定caspase及细胞小管形成实验观察NAV-LAAO对人脐静脉内皮细胞(HUVEC)生长和血管生成的影响。结果眼镜蛇毒粗毒经两步层析法得到的LAAO,分子量约为58 ku。NAV-LAAO抑制HUVEC细胞的生长和细胞小管形成,其抑制细胞生长的IC50为21.42 mg.L-1。与对照组相比,LAAO组caspase-3、caspase-8升高。结论应用两步层析法从眼镜蛇毒中分离出的NAV-LAAO具有剂量依赖性的抑制HUVEC细胞的生长和影响细胞小管形成的作用。     

舟山眼镜蛇毒细胞毒素的克隆及原核表达

目的构建舟山眼镜蛇毒细胞毒素（Cytotoxin,CTX）原核表达载体（pET42α（＋）-CTX）,并在大肠杆菌中表达CTX重组蛋白。方法从舟山眼镜蛇毒中分离纯化细胞毒素（CTX）,测定N-末端氨基酸序列并据此设计引物,以提取的舟山眼镜蛇毒腺总RNA为模板,通过RT-PCR方法扩增细胞毒素基因序列（CTXcDNA）。将CTXcDNA定向克隆到原核表达载体pET42α（＋）中,双酶切图谱、PCR和DNA测序鉴定,转化宿主菌E.coliBL21（DE3）,异丙基硫代-β-D半乳糖苷（IPTG）诱导,SDS-PAGE凝胶电泳分析表达产物,GSTrap FF亲和层析纯化融合蛋白CTX;Western-blotting鉴定。结果成功合成CTXcDNA,并构建原核表达质粒pET42α（＋）/GST-CTX,在大肠杆菌E.coliBL21（DE3）中表达融合蛋白CTX,SDS-PAGE凝胶浓度扫描显示表达量占菌体总蛋白的28.9%,Western-blotting证实纯化蛋白为重组CTX融合蛋白（rCTX）。结论成功构建CTX基因的原核表达载体,并在大肠杆菌中表达,获得rCTX具有眼镜蛇毒CTX的抗原性。     

舟山眼镜蛇毒心脏毒素-13的纯化、活性及尼群地平对其作用的影响

目的　从舟山眼镜蛇 (Najaatra)毒分离纯化心脏毒素并观察其心脏毒性作用和探讨作用机制。方法　用离子交换和凝胶过滤法从蛇毒中分离纯化心脏毒素 ,观察其对整体动物 ,离体器官和组织的作用及作用影响因素。结果　从舟山眼镜蛇毒中分离出 4个有心脏毒性组分 ,纯化其中 1个定名为心脏毒素 13(Cardiotoxin 13,CTX 13) ,它含有 6 0个氨基酸 ,分子质量 7 76 9ku。小鼠ivLD50 为 0 75 6mg·kg-1。对大鼠离体心脏CTX 13可使冠脉阻力增高 ,心脏收缩幅度减小 ,停于收缩期。尼群地平 (Nit)能取消CTX 13升高冠脉阻力作用 ,同时使CTX 13的负性肌力作用逆转为正性肌力作用。CTX 13可诱发猪离体冠脉环呈剂量依赖性收缩 ,其EC50 为 2 6 0mg·L-1,Nit 0 0 5 μmol·L-1使其量效曲线平行右移 ,EC50 提高到 36 0mg·L-1。但对于大鼠离体乳头肌Nit 0 83μmol·L-1对CTX 13所致心脏毒性无明显对抗作用。在整体实验中Ni0 3mg·kg-1可使大鼠ivCTX 13的最小致死量 (MLD)从 (44 4 7± 2 8 5 ) μg·kg-1提高到(5 41 2± 2 3 2 ) μg·kg-1。结论　CTX 13的心脏毒性作用除其直接心脏毒作用外 ,尚与其收缩冠脉作用有关     

眼镜蛇毒L-氨基酸氧化酶的纯化、性质鉴定及细胞毒性测定

目的从眼镜蛇蛇毒中分离L-氨基酸氧化酶（LAAO）,测定其理化性质及细胞毒性。方法采用Superdex75凝胶层析、Phenyl-SepharoseFF疏水层析、ResourceS离子交换层析分离纯化LAAO;SDS-聚丙烯酰胺凝胶电泳及C18反相色谱鉴定纯度并测定相对分子量;CCK8法测定细胞毒性。结果舟山眼镜蛇蛇毒经凝胶层析、疏水层析、离子交换层析及C18反相色谱后获得LAAO（暂定名NA-LAAO）,在非还原和还原条件下相对分子质量均为58kD左右;具有明显的抑制人胃癌MGC-803细胞增殖作用。结论从眼镜蛇毒中纯化得到LAAO,其具有明显的细胞毒性。     

眼镜蛇毒因子对实验性家犬呼吸窘迫综合征的保护作用

观察从中华眼镜蛇(Najanajaatra)蛇毒中分离纯化的眼镜蛇因子对油酸型呼吸窘迫综合征的保护作用。方法利用质谱仪、呼吸流量计和压力换能器分别连续监测麻醉狗的主要呼吸功能参数 ,实验后取肺组织作病理学检查。结果预先用足量眼镜蛇毒因子使体内补体耗竭95 %以上 ,可显著减轻油酸引起的肺功能障碍 ,组织学检查发现在透明膜形成、肺泡内出血及肺小动脉栓塞等方面均有显著改善。结论活化的补体在油酸引起的肺损伤的发展过程中发挥着重要作用 ,眼镜蛇毒因子通过耗竭血清补体可抑制呼吸窘迫综合征的发展目的观察从中华眼镜蛇(Najanajaatra)蛇毒中分离纯化的眼镜蛇因子对油酸型呼吸窘迫综合征的保护作用。方法利用质谱仪、呼吸流量计和压力换能器分别连续监测麻醉狗的主要呼吸功能参数 ,实验后取肺组织作病理学检查。结果预先用足量眼镜蛇毒因子使体内补体耗竭95 %以上 ,可显著减轻油酸引起的肺功能障碍 ,组织学检查发现在透明膜形成、肺泡内出血及肺小动脉栓塞等方面均有显著改善。结论活化的补体在油酸引起的肺损伤的发展过程中发挥着重要作用 ,眼镜蛇毒因子通过耗竭血清补体可抑制呼吸窘迫综合征的发展     

眼镜蛇毒蛋白酶natrahagin抑制兔颈动脉血栓形成的作用

眼镜蛇毒蛋白酶ｎａｔｒａｈａｇｉｎ是从中国眼镜蛇(Ｎａｊａｎａｊａａｔｒａ)毒中分离纯化出的蛋白酶,在体外可水解血小板膜糖蛋白Ⅰｂ(ＧＰⅠｂ)并具有抑制血小板聚集的作用[1]。研究表明,抑制ＧＰⅠｂ与其粘附蛋白配基之间相互作用的药物,能够抑制血小板的聚集与粘附反应,从而产生抗血栓的作用[2]。本文旨在观察ｎａｔｒａｈａｇｉｎ在体内对动脉血栓形成和血小板聚集的影响,为寻找新型抗血栓药物提供有价值的实验资料。1　材料和方法1.1　材料　ｎａｔｒａｈａｇｉｎ由第一军医大学药物研究所从中国眼镜蛇粗毒(由广州医学院蛇毒研究所…     

眼镜蛇毒分泌型磷脂酶A_2的研究进展

眼镜蛇毒分泌型磷脂酶A2(sPLA2)作为眼镜蛇毒中的一个重要成分,有着广泛的生物学活性,近年来的研究热点正日益从眼镜蛇粗毒转向毒素中的某个单体,如sPLA2的研究。该文简要综述了眼镜蛇毒sPLA2的结构特征、分离纯化以及分子生物学特性,着重论述了sPLA2多样的药理毒理作用,并对其作用机制和发展前景进行了探讨。     

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。     

Factors in snake venoms that increase capillary permeability

Capillary permeability increasing (CPI) activity is a phenomenon of the microvasculature caused by many agents such as snake venoms, histamine, 5-hydroxytryptamine (5-HT), prostaglandins and leukotrienes. Since no systematic study has been done to determine what components of snake venom cause CPI activity, a CPI factor from Naja naja atra (Taiwan cobra) venom was isolated using intravenous injections of Evan's blue dye as the indicator of increased permeability and the factor's properties were extensively studied. Cardiotoxin from Naja naja kaouthia (Thailand cobra) and Mojave toxin from Crotalus scutulatus scutulatus (Mojave rattlesnake) venoms demonstrated CPI activity. Postsynaptic neurotoxins from an elapid and a hydrophid and myotoxin a from Crotalus viridis viridis (prairie rattlesnake) showed no CPI activity at the dose studied. The purified CPI active component from Naja naja atra venom was found to have cardiotoxic activity. Therefore, Elapidae cardiotoxins are CPI active factors. However, CPI activity is not due to cardiotoxins alone as the presynaptic neurotoxin, Mojave toxin, also showed CPI activity. Selective inhibitors were used to indicate possible mechanisms of action on the capillaries by Naja naja atra venom and Crotalus scutulatus scutulatus venom. The histamine H1-receptor blockers diphenhydramine, promethazine, and cyproheptadine were effective against both venoms in preventing increased capillary permeability. These results suggested that histamine release activity is the most likely mechanism resulting in CPI activity from these venoms.     

Purification and characterization of a neurotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom.

Snake venoms contain multimolecular forms of phospholipase A2 which are diverse with respect to their pharmacological properties. A neurotoxic PLA2 from Naja naja naja venom has been purified in two steps. (1) The whole venom was fractionated on CM-Sephadex C-25 column; 4.6% of the total PLA2 activity recovered was found in the NN-V fraction. (2) The NN-Vb-PLA2 fraction was purified to homogeneity by gel filtration of fraction NN-V on Sephadex G-50. It is a basic protein with a mol. wt between 10,500 and 11,000, and is more toxic than other basic PLA2s purified from Naja naja naja venom. The LD50 of NN-Vb-PLA2 is 0.27 mg/kg body wt. It induced neurotoxic symptoms in experimental mice and is devoid of myotoxic, anticoagulant and edema-inducing activities.     

Antiflammins. Anti-inflammatory activity and effect on human phospholipase A2.

Two anti-inflammatory peptides (antiflammins) corresponding to a high amino acid similarity region between lipocortin I and uteroglobin were tested for their ability to inhibit purified human synovial fluid phospholipase A2 (HSF-PLA2). No inhibitory activity was observed, even at such high concentrations of peptides as 50 microM. When antiflammins were preincubated with the enzyme and/or the substrate, no HSF-PLA2 inhibition was detected. In vivo anti-inflammatory activity of these peptides was evaluated in several experimental models of inflammation induced by carrageenan, croton-oil, oxazolone and Naja naja naja venom phospholipase A2 (PLA2). In contrast to the in vitro results, anti-inflammatory activity was observed in all tests, except when inflammation was induced by snake venom PLA2. Taken together, our results do not support the hypothesis that the in vivo anti-inflammatory effect of antiflammins is directly related to inhibition of PLA2 activity.     

Interactions in red blood cells between fatty acids and either snake venom cardiotoxin or halothane

Phospholipase A2 (PLA2) activity enhances snake venom cardiotoxin (CTX)-induced and general anesthetic (halothane)-induced hemolysis of red blood cells. In the case of halothane-induced hemolysis, this effect appears to be related primarily to free fatty acids. In the present study, the interaction between CTXs and halothane and the effects of different free fatty acids on cardiotoxin and halothane-induced hemolysis were examined. The hemolytic actions of halothane and a CTX from Naja naja kaouthia venom were examined in erythrocytes with different phospholipid and free fatty acid composition from five species. The extent of hemolysis by CTX or halothane was dependent upon the species examined and appeared to be inversely related to the amount of saturated free fatty acid in the membrane. The order of susceptibility of red blood cells from five species to hemolysis was similar for halothane- and N. n. kaouthia CTX-induced hemolysis, but very different for osmotic fragility. The slope of the hemolysis dose-response curve was considerably steeper for halothane than for CTX. Hemolysis due to N. n. kaouthia CTX was greatly increased by halothane in erythrocytes from humans and horses and to a lesser extent in erythrocytes from sheep, goats and cows. Hemolysis induced by halothane and the N. n. kaouthia or Naja naja atra CTXs was enhanced by unsaturated fatty acids. In contrast, hemolysis induced by halothane was decreased and that caused by the two CTXs was relatively unaffected by saturated fatty acids. Halothane and CTXs differ in their exact mechanisms, but appear to act upon similar fatty acid-sensitive processes.     

一种水解血小板膜糖蛋白GPIb的眼镜蛇毒组分的分离纯化与鉴定

目的　建立从中华眼镜蛇毒中分离、纯化可水解血小板膜糖蛋白Ｉｂ（ｐｌａｔｅｌｅｔｍｅｍｂｒａｎｅｇｌｙｃｏｐｒｏｔｅｉｎＩｂ,ＧＰＩｂ）组分的方法。方法　肝素亲和层析,分子筛层析,ＳＤＳ聚丙烯酰胺凝胶电泳（ＳＤＳ ＰＡＧＥ）。结果　经Ｈｅｐａｒｉｎ ＳｅｈｐａｒｏｓｅＣＬ ６Ｂ亲和层析及ＳｅｐｈａｄｅｘＧ １５０分子筛层析,从中华眼镜蛇毒纯化出可降解纤维蛋白原和ＧＰＩｂ的活性组分,回收率为０－８８％,ＳＤＳ ＰＡＧＥ证实此组分为相对分子量约４７１００的单肽链蛋白。结论　此方法成功地从中华眼镜蛇毒中纯化出了水解ＧＰＩｂ的组分,具有高效、简便的特点。     

Effect of crotapotin and heparin on the rat paw oedema induced by different secretory phospholipases A2.

The effects of crotapotin (a non-toxic and non-enzymatic acid polypeptide naturally complexed with phospholipase A2) and heparin on rat paw edema induced by different secretory phospholipases A2 (sPLA2) have been investigated. The ability of crotapotin to affect the enzymatic activity of the sPLA2(s) have also been evaluated. Secretory PLA2(s) obtained from both snake (Naja naja, Naja mocambique mocambique, Crotalus adamanteus and Crotalus durissus terrificus) and bee (Apis mellifera) venoms as well as that from bovine pancreas were used in this study. Rat paw oedema was induced by a single subplantar injection of the sPLA2s (5-30 microg/paw) in absence and presence of either crotapotin (10-100 microg/paw) or heparin (50 U/paw). Paw volume was measured using a hydroplethysmometer. Phospholipase A2 from Naja naja, Naja mocambique mocambique, Apis mellifera venoms and the basic component of Crotalus durissus terrificus venom all induced dose-dependent rat paw oedema whereas those from Crotalus adamanteus venom and bovine pancreas were ineffective. Paw oedema induced by PLA2(s) from both Naja naja and Apis mellifera venoms was significantly (P < 0.05) inhibited by crotapotin (0.1-100 microg/site) whereas the Naja mocambique mocambique venom PLA2-induced oedema was significantly potentiated (P < 0.05) by this polypeptide (40 microg/site). On the other hand, heparin (50 U/paw) had no effect on the paw oedema induced by PLA2 from Naja naja and Apis mellifera venoms but significantly inhibited the Naja mocambique mocambique venom PLA2-induced oedema. The measurement of the in vitro phospholipasic activity revealed that crotapotin inhibited by 60-70% the enzymatic activities of PLA2(s) from Crotalus adamanteus, Naja mocambique mocambique, Apis mellifera venoms and bovine pancreas. Our results suggest that despite the great homology between the various types of sPLA2 they interact with crotapotin on cell surfaces in different ways leading to either inhibition or potentiation of the paw oedema by a mechanism unrelated to their enzymatic activities. Since heparin reduced paw oedema induced by PLA2 from Naja mocambique mocambique venom it is likely that this sPLA2 is similar to the novel heparin-sensitive PLA2 found in mast cells.     

Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A2 by modification of carboxylate groups

The carboxylate groups in an acidic and in a basic phospholipase A2 (PLA2) enzyme, purified, respectively, from Naja naja atra and Naja nigricollis snake venoms, were modified with carbodiimide and semicarbazide. The derivatives modified at pH 3.5 and pH 5.5 had less than 1% (N. nigricollis) or 2% (N. n. atra) residual enzymatic activity, whereas 12-16% enzymatic activity remained following modification at pH 5.5 in the presence of Ca2+. In marked contrast, these derivatives retained variable, but significantly greater, levels of lethal potency, hemolytic and anticoagulant activities, and abilities to block indirectly and directly induced contractions of the diaphragm. By this modification of aspartic and glutamic acid residues we have, for the first time, obtained derivatives of PLA2 which selectively retain greater pharmacological activity relative to enzymatic activity. Previously, we had found that modification of lysine and arginine residues produced derivatives which retain enzymatic activity but show a loss of pharmacological properties. These findings suggest that some pharmacological effects of snake venom PLA2 enzymes are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.