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
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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-6 B 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.
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-6 B 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.
引文
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[15] 孙林,张学荣,蔡凤桃,等.眼镜蛇毒细胞毒素-4N的分离纯化及其对HSC-T6细胞的作用[J].基因组学与应用生物学,2017,36(6):2313-2319.
[2] Gan F,Liu Q,Liu YH,et al.Lycium barbarum polysaccharides improve CCl4-induced liver fibrosis,inflammatory response and TLRs/NF-kB signaling pathway expression in wistar rats[J].Life Sci,2018,192:205-212.
[3] Friedman SL.Mechanisms of Hepatic Fibrogenesis[J].Gastroenterology,2008,134(6):1655-1669.
[4] Bai FC,Huang QF,Nie JL,et al.Trolline Ameliorates Liver Fibrosis by Inhibiting the NF-κB Pathway,Promoting HSC Apoptosis and Suppressing Autophagy[J].Cell Physiol Biochem,2017,44:436-446.
[5] Cheng Y,Zheng H,Wang B,et al.Sorafenib and fluvastatin synergistically alleviate hepatic fibrosis via inhibiting the TGFβ1/Smad3 pathway[J].Dig Liver Dis,2018,50(4):381-388.
[6] Zhang J,Yang A,Wu Y,et al.Stachydrine ameliorates carbon tetrachloride-induced hepatic fibrosis by inhibiting inflammation,oxidative stress and regulating MMPs/TIMPs system in rats[J].Biomed Pharmacother,2018,97:1586-1594.
[7] 赵健楠,孙晋民.蛇毒抗肿瘤成分的研究进展[J].西北药学杂志,2017,32(3):391-393.
[8] 张迎征,和七一,邓秋平,余晓东.眼镜蛇属(Naja)毒液的主要功能组分研究进展[J].延安大学学报(自然科学版),2016,35(1):49-54.
[9] Tsai PC,Chu CL,Chiu CC,et al.Inhibition of Src activation with cardiotoxin III blocks migration and invasion of MDA-MB-231 cells[J].Toxicon,2013,74:56-67.
[10] Tsai PC,Chu CL,Chiu CC,et al.Cardiotoxin III suppresses hepatocyte growth factor-stimulated migration and invasion of MDA-MB-231 cells[J].Cell Biochem Funct,2014,32(6):485-495.
[11] Yang SH,Chien CM,Lu MC,et al.Cardiotoxin III induces apoptosis in K562 cells through a mitochondrial-mediated pathway[J].Clin Exp Pharmacol Physiol,2005,32(7):515-520.
[12] Chien CM,Yang SH,Yang CC,et al.Cardiotoxin III induces c-jun N-terminal kinase-dependent apoptosis in HL-60 human leukaemia cells[J].Cell Biochem Funct,2008,26(1):111-118.
[13] Wu MY,Ming W,Tang Y,et al.The anticancer effect of cytotoxin 1 from Naja atra Cantor venom is mediated by a lysosomal cell death pathway involving lysosomal membrane permeabilization and cathepsin B release[J].Am J Chin Med,2013,41(3):643-663.
[14] 蒋世强,潘能庆,鄢航,等.CTX研究进展[J].甘肃畜牧兽医,2017,47(3):26-28.
[15] 孙林,张学荣,蔡凤桃,等.眼镜蛇毒细胞毒素-4N的分离纯化及其对HSC-T6细胞的作用[J].基因组学与应用生物学,2017,36(6):2313-2319.
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