眼镜蛇细胞毒素致昆明小鼠局部皮肤坏死模型的构建  

A Kunming mice local skin necrosis model of the cytotoxin of Chinese cobra

作　　者：陆雪玲 朱俊蓉 黄周 黄东凌 王璠 韦晚霞 刘恋 王威[1] LU Xueling;ZHU Junrong;HUANG Zhou;HUANG Dongling;WANG Fan;WEI Wanxia;LIU Lian;WANG Wei(Department of Emergency,the First Affiliated Hospital Guangxi Medical University,Nanning City,Guangxi Zhuang Autonomous Region,530021,China;Guangxi University Key Laboratory of Emergency Medicine,Nanning City,Guangxi Zhuang Autonomous Region,530021,China)

机构地区：[1]广西医科大学第一附属医院急诊科,广西南宁530021 [2]广西高校急诊医学重点实验室,广西南宁530021

出　　处：《蛇志》2024年第1期6-11,共6页Journal of Snake

基　　金：区域创新合作项目(项目名称:基于虚拟标准化蛇伤患者治疗模型的蛇伤大数据平台研究及示范应用,项目编号:2022YFQ0085)。

摘　　要：目的通过皮内注射中华眼镜蛇细胞毒素(CTX)建立中华眼镜蛇咬伤皮肤坏死小鼠模型。方法通过凝胶层析+离子层析从中华眼镜蛇粗毒中分离出眼镜蛇细胞毒素(CTX-X)。取30只小鼠随机分为10组:生理盐水组、10μg粗毒组、20μg粗毒组、25μg粗毒组、10μg CTX-X组、20μg CTX-X组、30μg CTX-X组、40μg CTX-X组、50μg CTX-X组、60μg CTX-X组,每组3只。注毒72 h后观察小鼠注射部位皮肤坏死直径及病理变化。结果(1)中华眼镜蛇粗毒通过Sephadex G-50凝胶层析分离获得2个蛋白峰(F1、F2),将CTX-X所在的F2峰经过CM Sepharose TM CL-6B阳离子交换柱色谱分离获得F1、F2、F3、F44个峰,通过小鼠体内实验及SDS-PAGE确定F4峰分离的CTX-X具有活性且达到电泳纯。(2)CTX-X组小鼠的皮内坏死直径随着CTX-X剂量增加而增大,51.21μg CTX-X组的小鼠皮内坏死直径均值达到5 mm。粗毒组小鼠随着皮内注射粗毒剂量的增加,小鼠72 h内的死亡率增高,当皮内注射粗毒剂量<25μg时,注毒72 h后处死并解剖小鼠,注射部位未观察到皮肤坏死情况;当皮内注射粗毒剂量≥25μg时,所有小鼠在注毒后3 h内死亡,且皮内注射部位未见明显坏死。结论应用Sephadex G-50凝胶过滤+CM Sepharose TM CL-6B阳离子交换分离所得的CTX-X,于小鼠背部皮内注射可成功构建中华眼镜蛇CTX-X致局部皮肤坏死的动物模型,该模型稳定性高且具备良好的可复制性。Objective To establish a Kunming mice's local skin necrosis model by intradermal injection of Chinese cobra's cytotoxin(CTX).Methods Cobra cytotoxin(CTX-X)was isolated from the crude venom of Chinese cobra by gel chromatography+ion chromatography.30 mice were randomly divided into 10 groups:Normal saline group,10μg crude venom group,20μg crude venom group,25μg crude venom group,10μg CTX-X group,20μg CTX-X group,30μg CTX-X group,40μg CTX-X group,50μg CTX-X group,60μg CTX-X group,and 3 rats in each group.Observe the diameter of skin necrosis and pathological changes at the injection site for 72 h.Results(1)The crude cobra venom was separated by Sephadex G-50 gel chromatography to obtain two protein peaks(F1,F2),and the second peak(F2)where the CTX-X was located was separated by CM SepharoseTMCL-6B cation-exchange column chromatography to obtain four peaks,including F1,F2,F3,and F4.CTX-X activity was determined by in vivo mouse assay and CTX-X purity was determined by SDS-PAGE.(2)The diameter of intradermal necrosis in the CTX-X group of mice increased with increasing CTX-X dose,and the mean intradermal necrosis diameter in the 51.21μg CTX-X group reached 5mm;In the crude group,the mortality rate of mice within 72 h increased with the increase of the intradermal injection dose of crude poison.When the intradermal injection dose of crude poison was<25μg,mice were executed and dissected after 72 h of injection,no skin necrosis was observed at the injection site,and when the intradermal injection dose of crude poison was≥25μg,all the mice were dead within 3 h of injection,and no obvious necrosis was observed at the intradermal injection site.Conclusion A mice model of CTX-X-induced local skin necrosis in Chinese cobras was successfully constructed by intradermal injection of CTX-X obtained by Sephadex G-50 gel filtration+CM SepharoseTMCL-6B cation exchange,which was highly stable and reproducible.

关 键 词：中华眼镜蛇 细胞毒素 皮肤坏死 动物模型 

分 类 号：R996.3[医药卫生—毒理学]