人单核细胞株THP-1中HMGN2的分离纯化及其抗乙型肝炎病毒活性  

Isolation and purification of antimicrobial polypeptide HMGN2 from human monocyte cell line THP-1 and detecting of its antiviral activity against hepatitis B virus

作　　者：张敏[1] 张远[1] 童荣生[1] 

机构地区：[1]四川省医学科学院.四川省人民医院药剂科,四川成都610072

出　　处：《中国医院药学杂志》2014年第17期1445-1449,共5页Chinese Journal of Hospital Pharmacy

基　　金：国家自然科学基金项目(编号:30300127)

摘　　要：目的：从人单核细胞株THP-1中分离与纯化HMGN2抗菌肽，应用HepG2．2．15细胞株作为体外抗乙型肝炎病毒的实验模型，以探讨HMGN2分子的抗HBV作用。方法：采用HPLC分离纯化，Tricine-SDSPAGE电泳、质谱精确分子量测定、Westernblotting对分离纯化得到的HMGN2分子进行分子鉴定。以HBVDNA转染的细胞株HepG2．2．15细胞为模型，用不同浓度HMGN2分子作用于HepG2．2．15细胞，分别在第4天和第8天收集细胞培养上清液，ELISA检测上清中HBV表面抗原（HBsAg）及e抗原（HBeAg），采用实时荧光定量PCR法检测上清液HBVDNA的含量。结果：从人单核细胞株THp-1中分离纯化获得纯度较高的HMGN2蛋白；其在1-100mg·L-1范围内，对HepG2．2．15细胞无细胞毒性；HMGN2蛋白分子在1-5mg·L-1水平即可显著抑制HBeAg和HBsAg的表达，可显著降低HBVDNA拷贝数，继续增加剂量抗病毒效果不再明显增加。结论：HMGN2在体外细胞培养中具有直接抗HBV活性。OBJECTIVE To investigate the anti HBV effect of HMGN2 through isolation and purification of HMGN2 peptide from human monocytic cell line THP-1 and utilization of HepG 2. 2. 15 cell line as in vitro model for anti-hepatitis B virus research. METHODS HMGN2 was isolated and purified by HPLC. Tricine-SDS-PAGE electrophoresis, Western blotting and mass spectrometry were used for identification of this molecule. Different concentrations of HMGN2 were applied to HepG 2. 2.15 cell line, and the supernatant of 4 d and 8 d were collected for investigation. ELISA was used to detect HBsAg and HBeAg, and real-time PCR was used to detect the HBV DNA level. RESULTS High level of purified protein was obtained from human monocytic cell line THP-1, and the detective range was in 1-100 mg· L-1. No cytotoxic effect of HMGN2 was observed in HepG 2. 2. 15, and HMGN2 could obviously suppress the expression of HBeAg and HBsAg, and lowered the HBVDNA copy number. The anti-virus effect would not incre.ase when the dosage of HMGN2 was increased. CONCLUSION It was showed that HMGN2 had direct anti-HBV activity in vitro.

关 键 词：人THP-1细胞 HMGN2 分离纯化 抗HBV 

分 类 号：R969[医药卫生—药理学]