僵蚕溶茧酶抑制剂的纯化与抗肿瘤活性研究  

Research on purification of cocoonase inhibitor of Bombyx Batryticatus and its anti-tumor activity

作　　者：王厚伟[1] 徐凌川[1] 王家超 曾英姿 田景振[1] 窦彦玲[2] 王飞 WANG Hou-wei;XU Ling-chuan;WANG Jia-chao;ZENG Ying-zi;TIAN Jing-zhen;DOU Yan-ling;WANG Fei(School of Pharmacy,Shandong University of Traditional Chinese Medicine,Jinan 250355,China;School of Intelligence and Information Engineering,Shandong University of Traditional Chinese Medicine,Jinan 250355,China;School of Basic Medicine,Hebei Medical University,Shijiazhuang 050017,China;Shandong Wohua Pharmaceutical Technology Co.,Ltd.,Weifang 261061,China;Yuhua Jingchen(Shandong)Health Development Group Co.,Ltd.,Jinan 250000,China)

机构地区：[1]山东中医药大学药学院,山东济南250355 [2]山东中医药大学智能与信息工程学院,山东济南250355 [3]河北医科大学基础医学院,河北石家庄050017 [4]山东沃华医药科技有限公司,山东潍坊261061 [5]御华景宸(山东)康养发展集团有限公司,山东济南250000

出　　处：《中草药》2022年第7期2022-2030,共9页Chinese Traditional and Herbal Drugs

基　　金：山东省自然科学基金资助项目(ZR2013HM035);山东省重点产业关键技术资助项目(2016CYJS08A01-8);济南科技发展计划资助项目(201102021);山东省高校科技计划资助项目(J11LF29)。

摘　　要：目的分离纯化僵蚕溶茧酶抑制剂(Jiangcan cocoonase inhibitor,JCCI),研究其体内外对人肝癌SMCC-7721细胞增殖的抑制活性。方法依次应用以家蚕溶茧酶为配体的亲和色谱、Sephadex G-50凝胶过滤色谱、Superdex 75快速蛋白液相色谱(fast protein liquid chromatography,FPLC),从僵蚕粗蛋白提取物的85%硫酸铵沉淀物中分离纯化JCCI。以Edman降解法测其N端氨基酸序列。应用MTT法与荷瘤裸鼠模型,检测其体内外抑制SMCC-7721细胞增殖与生长活性。结果JCCI相对分子质量为13973.63,其N端前10个氨基酸序列为VRNKRQSNDD。抑制剂动力学分析结果表明,JCCI是溶茧酶的非竞争性抑制剂,米氏常数(Km)平均值为76.50,二者物质的量之比为1∶1。JCCI可显著抑制SMCC-7721肝癌细胞体外增殖与体内荷瘤裸鼠的肿瘤生长,体外给药36 h的半数抑制浓度(median inhibition concentration,IC_(50))为260.52μg/mL,JCCI抑制率与剂量线性相关。结论JCCI是一种首次从僵蚕中分离纯化的具有抗肿瘤活性的丝氨酸蛋白酶抑制剂。Objective To isolate and purify the cocoonase inhibito of Bombyx Batryticatusr(Jiangcan,JCCI),and study its anti-tumor activity of SMCC-7721 liver cancer cells in vitro and in vivo.Methods JCCI was purified from the 85%ammonium sulfate precipitate of the crude protein extract of Bombyx Batryticatus by affinity chromatography with silkworm cocoonase as ligand,Sephadex G-50 gel filtration chromatography,and Superdex 75 FPLC.The JCCI N-terminal amino acid sequence was determined by Edman degradation method.MTT method and tumor-bearing nude mouse model were used to detect the inhibiting effect of JCCI on the proliferation and growth of SMCC-7721 in vivo and in vitro.Results The molecular weight of JCCI was 13973.63 Daltons,and the first 10 amino acid sequence of its N-terminal was VRNKRQSNDD.JCCI was a non-competitive cocoonase inhibitor,with an average Km of 76.50 and a molar inhibition ratio of 1∶1.JCCI could significantly inhibit the proliferation of SMCC-7721 in vitro and the tumor growth of tumor-bearing nude mice in vivo.The IC_(50) was 260.52μg/mL after 24 h administration in vitro,and the inhibition rate was linearly related to the dose of JCCI.Conclusion JCCI was a serine protease inhibitor with anti-tumor activity purified from Bombyx Batryticatus for the first time.

关 键 词：僵蚕 溶茧酶 丝氨酸蛋白酶抑制剂 抗肿瘤活性 肝癌 亲和色谱 荷瘤裸鼠 MTT法 

分 类 号：R284.1[医药卫生—中药学]