广西眼镜蛇毒细胞毒素-2的分离纯化及其对大鼠肝星状细胞HSC-T6的作用  

Separation and Purification of Cobra Venom Cytotoxin-2 and its Inhibitory Action on HSC-T6

作　　者：王秀男 张学荣 廖明 班建东 农君 陶慧娟 陈荣芳 WANG Xiu-nan;ZHANG Xue-rong;LIAO Ming;BAN Jian-dong;NONG Jun;TAO Hui-juan;CHEN Rong-fang(School of Preclinical Medicine,Guangxi Medical University,Nanning City,Guangxi Zhuang Autonomous Region,530021,China;Life Sciences Institute,Guangxi Medical University,Nanning City,Guangxi Zhuang Autonomous Region,530021,China)

机构地区：[1]广西医科大学基础医学院,广西南宁530021 [2]广西医科大学生命科学研究院,广西南宁530021

出　　处：《蛇志》2019年第1期1-4,共4页Journal of Snake

基　　金：国家自然科学基金资助项目(项目编号:81360078;81860344)

摘　　要：目的通过层析法从广西眼镜蛇毒中分离得到电泳纯的细胞毒素-2(CTX-2),探索CTX-2对大鼠肝星状细胞(HSC-T6)的增殖抑制作用。方法采用DEAE-Sepharose CL-6B阴离子交换层析、Spehadex G-50凝胶层析和Macro-prep High S阳离子交换层析结合的方法分离眼镜蛇毒粗毒;经SDS-PAGE电泳鉴定蛋白纯度;NanoLC-ESI-MS/MS质谱方法鉴定其组分并测定分子量;CCK-8法检测CTX-2对HSC-T6的增殖抑制作用,确定其最小有效浓度。结果眼镜蛇毒粗毒经分离纯化获得电泳纯的CTX-2,其分子量约为9.548 kD;不同浓度CTX-2作用于HSC-T6细胞24 h后,其增殖抑制作用随浓度增加而增大,呈量效关系,抑制增殖的最小有效浓度为8 mg/L,IC_(50)为11.52 mg/L。结论广西眼镜蛇毒粗毒经三步分离法得到电泳纯且具有高生物活性的CTX-2;广西眼镜蛇毒CTX-2可抑制HSC-T6细胞增殖,抑制作用呈量效关系。Objective To purify cytotoxin-2 (CTX-2) of the electrophoretically purity from the cobra venom by chromatography and to study its inhibitory action on HSC-T6 cells.Methods The CTX-2 was purified separately by DEAE-Sepharose CL-6B ion exchange chromatography,Spehadex G-50 molecular sieve chromatography and Macro-prep High S ion exchange chromatography.The purity of end-product was identified via SDS-PAGE electrophoresis,and its compositions were identified by NanoLC-ESI-MS / MS mass spectrometry.CCK-8 method was used to detect the proliferation of HSC-T6 cells with different concentrations of CTX-2 to find out the minimum effective concentration.Results The CTX-2 of the electrophoretically purity was purified by chromatography with a molecular weight of 9.548 kD.After 24 hours of treatment with different concentrations of CTX-2 on HSC-T6 cells,the inhibition of proliferation increased with increasing concentration,showing a dose-effect relationship.The minimum effective concentration of CTX-2 to HSC-T6 cells was 8 mg/L,and the IC50 was 11.52 mg/L.Conclusion Electrophoretic pure CTX-2 with high bioactivity was obtained by three-step separation method,and it can significantly inhibited the growth of HSC-T6 cells in a concentration dependent manner.

关 键 词：细胞毒素-2 肝纤维化 眼镜蛇毒 分离纯化 肝星状细胞 

分 类 号：R392.1[医药卫生—免疫学]