梁桓熙 王子依 李晶哲 马琰岩 刘长振 孙震晓

摘要 目的：对毛蚶抗癌蛋白（ASAP）进行了分离纯化，并研究分离后毛蚶抗癌蛋白主要组分的抗癌作用。方法：依次利用阴离子交换层析、疏水作用层析、凝胶过滤层析对ASAP进行分离;联合p53基因表达激活及CCK-8细胞活力测定检测分离组分的抗癌活性;CCK-8细胞活力测定法检测毛蚶抗癌蛋白主要组分对人结直肠癌Colo205、HT-29、人肺癌A549、人慢性髓性白血病K562细胞活力的影响;Annexin-V/PI双染结合流式细胞术检测细胞凋亡;Western Blotting法检测细胞凋亡相关蛋白：Mcl-1、Bcl-2、Bax、procaspase-3的表达情况。结果：从ASAP中分离得到的毛蚶抗癌活性蛋白主要组分C6在24～72 h内对Colo205、HT-29、A549、K562的细胞活力均有不同程度的抑制作用，其中对Colo205细胞的抑制作用最明显，作用72 h时IC50为5.7 μg/mL;C6可以诱导Colo205细胞凋亡且呈一定剂量依赖性;C6可以抑制抗凋亡蛋白Mcl-1和Bcl-2的表达，上调凋亡蛋白Bax的表达，引起凋亡蛋白caspase-3前体procaspase-3下降。结论：毛蚶抗癌蛋白主要组分C6对人多种癌细胞活力具有抑制作用，可诱导人结直肠癌Colo205细胞发生caspase通路依赖的细胞凋亡。

关键词 毛蚶抗癌蛋白主要组分C6;层析分离;Colo205;HT-29;A549;K562;细胞凋亡;Caspase

Abstract Objective：In this study，the anticancer protein（ASAP） of the larvae was isolated and purified，and the anticancer effects of the main components of the anticancer protein of the larvae were studied.Methods：We separated ASAP by anion exchange chromatography，hydrophobic interaction chromatography，and gel filtration chromatography; combined p53 gene expression activation and CCK-8 cell viability assay to track the anticancer activity of the separated components; the CCK-8 cell viability assay detected the effects of the main components of the anti-cancer protein of ASAP on the viability of human colorectal cancer Colo205，HT-29，human lung cancer A549，and human chronic myelogenous leukemia K562; Annexin-V/PI double staining combined with flow cytometry was used to detect cell apoptosis.Results：C6，the main component of the anticancer active protein isolated from ASAP，can inhibit the cell viability of Colo205，HT-29，A549，and K562 to varying degrees within 24 to 72 hours.Among them，it has the most inhibitory effect on Colo205 cells.Obviously，the IC50 was 5.7 μg/mL at 72 h; C6 can induce Colo205 cell apoptosis in a dose-dependent manner; C6 can inhibit the expression of anti-apoptotic proteins Mcl-1 and Bcl-2，and up-regulate the apoptotic protein Bax expression and cause the decrease of the apoptotic protein caspase-3 precursor procaspase-3.Conclusion：C6，the main component of the anti-cancer protein of ASAP，has an inhibitory effect on the viability of a variety of human cancer cells，and can induce caspase pathway-dependent apoptosis in human colorectal cancer Colo205 cells.

Keywords Main component of ASAP C6; Chromatographic separation; Colo205; HT-29; A549; K562; Apoptosis; Caspase

中图分类号：R73-36+2文獻标识码：Adoi：10.3969/j.issn.1673-7202.2021.20.009

毛蚶（Arca subcrenata Lischke）为软体动物门瓣鳃纲列齿目蚶科毛蚶属海洋生物，其药用历史悠久，其壳为中药瓦楞子（Arcae Concha）来源之一[1]，《医林纂要·药性》记载其能“攻坚破瘀。去一切痰积，血积，气块，破症瘕，攻瘰疬”，据《食疗本草》记载，其肉有“利五脏、温中、消食”等功效。

本课题组前期进行了毛蚶水提物体外抗癌作用筛选和抗癌蛋白的分离、纯化及抗癌机制……