摘要
目的:通过层析法从尖吻蝮蛇蛇毒中分离纯化降纤酶,探索降纤酶经直肠给药进入血液循环而降低体内纤维蛋白原水平的可行性.方法:从尖吻蝮蛇蛇毒粗毒中分离降纤酶,经小鼠直肠给药后动物成像观察降纤酶在小鼠脏器中的分布,病理学检测降纤酶对组织的影响,质谱测定小鼠血浆成分,ELISA检测血浆纤维蛋白原水平.结果:尖吻蝮蛇蛇毒粗毒分离纯化获得电泳纯的降纤酶,经直肠给药后其安全性高,无急性毒性反应,直肠组织无明显病理改变.免疫组化结果显示降纤酶分布于直肠组织中(P＜0.01),Shotgun质谱测得给药1h后小鼠血浆存在降纤酶片段,动物成像显示给药3 h后染料标记的降纤酶分子分布大量分布于盲肠组织中,并随时间的延长其含量逐步减少.降纤酶经直肠给药能降低体内纤维蛋白原水平(P＜0.01).结论:尖吻蝮蛇蛇毒粗毒经二步层析法分离获得电泳纯的降纤酶;降纤酶可经直肠给药后吸收进入血液循环并降低体内纤维蛋白原水平.
Abstract
OBJECTIVE To isolate and purify defibrase from agkistrodon venom by chromatography and explore the feasibil-ity of transrectal dosing into blood circulation to reduce the level of fibrinogen in vivo.METHODS Defibrase was isolated from crude venom of Agus agicoides.After a transrectal injection,the distribution of defibrase in the organs of mice was observed by animal imaging.The effect of defibrase on tissues was detected by pathology examination.RESULTS Electrophoretically pure defibrillinase was acquired by isolation and purification of crude venom of sharp-nosed pit viper.It was safe after transrectal dosing without acute toxicity or obvious pathological changes in rectal tissues.Immunohistochemistry indicated that fibrinogenin was dis-tributed in rectal tissues(P＜o.01).Shotgun mass spectrometry was utilized for measuring the plasma level of fibrinogen frag-ments after 1 h of dosing.Animal imaging revealed that dye-labeled fibrinogen was predominantly distributed in cecal tissues after 3 h of dosing with a gradual decrease.Rectal administration of fibrinogen reduced fibrinogen level in vivo(P＜0.01).CONCLUSION Electrophoretic pure defibrase is acquired from crude venom of agkistrodon by two-step chromatography.Defi-brase may be absorbed into blood circulation and reduce fibrinogen level after transrectal dosing.
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