中介蝮蛇毒组织损伤及粗毒抗血清的制备

发布时间：2018-02-11 18:13

本文关键词： 中介蝮组织损伤生物效应抗血清中毒保护　出处：《陕西师范大学》2011年硕士论文　论文类型：学位论文

【摘要】：中介蝮(Gloudius intermedius)属蝰科(Viperidae)蝮亚科(Crotalinae)亚洲蝮属(Gloydius),广泛分布于我国西北、华北部分地区,是我国6种蝮蛇中毒性最强的一种,它与分布区人民生活密切相关。蛇毒(snake venom)是蛇毒腺分泌的多种组分构成的复杂混合物,分别作用于不同的靶分子／靶细胞,可能造成多种中毒效应,包括肌肉组织损伤、出血、水肿、血压下降等。早在1979年,赵尔宓等人的研究发现,国产抗蝮蛇血清对中介蝮蛇毒没有中和作用,发现中介蝮的蛇毒组成与其它5种蝮蛇(短尾蝮(G. brevicaudus)、高原蝮(G. strauchii)、黑眉蝮(G. saxitilis)、乌苏里蝮(G. ussuriensis)及蛇岛蝮(G. shedaoensis)〕蛇毒相比有较大差异。由于中介蝮在北方分布广泛,危害大,目前还没有中介蝮蛇毒组织损伤方面的资料,加之缺乏特异性抗血清,这妨碍了蛇伤的有效治疗。研究内容及方法： (1)中介蝮粗毒对小鼠的毒理学效应及组织损伤,包括小鼠爪肿胀实验,粗毒皮下注射小鼠后,对肌肉组织(心肌、骨骼肌)损伤的形态学观察,大鼠尾静脉注射引起肌肉损伤后肌酸激酶(CK)释放检测,兔耳静脉注射后对血压的影响； (2)抗蛇毒血清的制备及中毒保护效果,包括将中介蝮粗毒与弗氏佐剂混合乳化,将其作为抗原免疫新西兰大白兔(初次免疫、5次加强免疫)后,收集兔抗血清,用免疫双向扩散法和间接ELISA法检测抗血清的滴度和效价,Western blot检测抗原结合特异性； (3)抗血清的中毒保护作用：将制备的抗血清按一定比例分别与致死剂量的蛇毒在体外混合孵育后,腹腔注射小鼠(体重18-20g),24h后,根据小鼠存活率,计算抗血清对中介蝮蛇毒的解毒效应。结果及讨论： (1)小鼠注射中介蝮蛇毒1.5小后,发现小鼠爪部开始有肿胀,3小时后出现明显肿胀； (2)与对照组相比,小鼠腹腔注射中介蝮蛇毒12h后,骨骼肌中有部分肌原纤维被水解破坏,肌纤维排列无序,肌浆有溶解现象；心肌纤维排列无序,部分肌丝出现溶解,肌原纤维间隙加大,出现明显损伤。 (3)尾静脉注射中介蝮蛇毒后,在3h-6h CK活力逐渐上升,6h达最高(1.174U/mL),是注射前的7.2倍,之后逐渐下降；注射蛇毒后,血压90min后呈快速下降趋势,至120min时,血压已降至给药前的40%以下,与同期生理盐水组相比差异非常显著(P0.01),这与国内已经报道的中介蝮咬伤人后的中毒症状相符。(4)琼脂双向免疫扩散试验显示,抗体1：2、1：4、1：8倍稀释后,可见明显免疫沉淀线,表明在免疫接种第十五周时,兔血液中已经有高滴度的抗血清产生。 (5)ELISA检测后发现,抗血清的有效效价不低于1：128000；而抗体Western blotting检测结果显示,阳性反应条带主要针对分子量14.4kD-18.4kD之间、41kD部位、70kD左右3组毒素部位,表明主要毒素蛋白刺激免疫系统产生了相应的抗体。 (6)在体外中和后,兔抗血清为0.2mL以上时,对0.76mg/kg中介蝮蛇毒有明显的保护作用,一半以上的受试小鼠存活。可见抗血清能明显延迟小鼠死亡时间,有解毒作用。结论：本研究所得结果为将来开发抗中介蝮蛇毒血清,用于蛇伤治疗奠定了基础。
[Abstract]:G.intermedius (Gloudius intermedius) (Viperidae) belongs to Viperidae Agkistrodon subfamily (Crotalinae) Gloydius (Gloydius), is widely distributed in Northwest China, North China, is one of 6 most toxic viper in our country, and it is closely related to the distribution of people's life in the area. The snake venom (snake venom) is the complex mixture of various groups of snake venom gland secretion, respectively for the target molecules / different target cells, may cause a variety of toxic effects, including muscle tissue injury, hemorrhage, edema, blood pressure decline.
In early 1979, Zhao Ermi et al study found that the domestic anti venom serum NO neutralization of venom, venom composition found intermedius and other 5 species of Agkistrodon brevicaudus (G. (brevicaudus) (G. strauchii), Agkistrodon strauchii (G. saxitilis), Agkistrodon saxatilis, Ussuri mamushi (G. and ussuriensis) Gloydius shedaoensis (G. shedaoensis) are quite different: compared to snake venom.
Because of g.intermedius wide distribution in northern China, the harm is great, there is no aspect of the venom tissue injury, and the lack of specific antiserum, which hinders the effective treatment of snakebites.
Research contents and methods:
(1) the toxicological effects of g.intermedius venom in mice and tissue injury, including mice paw swelling experiment, mice after subcutaneous injection of venom, muscle tissue (myocardium, skeletal muscle injury) to observe morphology, rat tail vein injection induced muscle damage after creatine kinase (CK) release assay, the effect on blood pressure after the injection of rabbit ear vein;
(2) the effect of preparation and poisoning protection of antivenom, including g.intermedius venom mixed with Freund's adjuvant emulsion, as antigen to immunize rabbits (initial immunization, 5 immunization) after collecting rabbit antiserum by double immune diffusion method and indirect ELISA method to detect the titer of antiserum and the titer of Western, detection of blot antigen binding specificity;
(3) poisoning the protective effects of antisera: the antiserum prepared according to a certain proportion respectively with a lethal dose of venom in vitro after incubation, intraperitoneal injection of mice (weight 18-20g), according to 24h, the survival rate of mice, calculation of detoxification effect on venom antiserum.
Results and discussions:
(1) after 1.5 small mice injected with Agkistrodon hutkkistrodon venom, it was found that the mouse claw began to swollen, and the swelling was obvious after 3 hours.
(2) compared with the control group, intraperitoneal injection of venom 12h, skeletal muscle in part by hydrolysis of myofibrillar damage, muscle fibers arranged disorderly, sarcoplasmic dissolution phenomenon; myocardial fibers arranged in disorder, part of myofilament dissolved, myofibril gap increased, the obvious damage.
(3) intravenous injection of venom increased gradually after the intermediary, the activity of CK in 3h-6h, 6h (1.174U/mL), the highest is 7.2 times before the injection, then decreased gradually; after the injection of venom, blood pressure decreased rapidly after 90min, to 120min, the blood pressure has dropped before administration of 40%, compared to compared with the normal saline group was significant difference (P0.01), and the symptoms of poisoning have been reported in the domestic g.intermedius bite after the match. (4) showed that agar double immunodiffusion test, 1:2,1:4,1:8 antibody dilution after immunoprecipitation showed that, at the fifteenth week after immunization, the rabbit blood has antiserum high titer.
(5) ELISA was found, the titer of antiserum effectively not less than 1:128000; and Western blotting antibody test results showed that the positive band mainly between molecular weight 14.4kD-18.4kD 41kD 70kD site, about 3 sets of toxin sites, showed that the main toxin stimulates the immune system to produce the corresponding antibody.
(6) in vitro after neutralization of rabbit antiserum is above 0.2mL, has obvious protective effect on 0.76mg/kg venom, more than half of the tested mice survival. Visible antiserum could obviously delay the death time of mice, detoxification.
Conclusion: the results of this study for the future development of anti venom serum for snakebite treatment laid the foundation.

【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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