HBIG抑制HBsAg、HBV DNA分泌的体外实验研究

Inhibition the secretion of HBsAg and HBV DNA by hepatitis Bimmunoglobulin in vitro

目的研究乙型肝炎免疫球蛋白（HBIG）对肝细胞的跨膜转运作用和对乙型肝炎病毒（HBV）感染细胞模型的乙型肝炎表面抗原（HBsAg）、HBVDNA分泌的抑制作用。方法用含HBIG的培养基培养QSG7701细胞，在不同时间点定量检测培养上清的抗HBs，计算HBIG的透过率；用含HBIG的培养基培养HepG2．2．15细胞数天，或先以，定浓度的HBIG共培养，第3天后更换为不含HBIG的培养基继续培养细胞。于不同时间点定量检测培养上清的HBsAg、HBV DNA；用MTT比色试验观察药物的细胞毒性。结果浓度为0.1～0.4IU／mL的HBIG与QSG7701细胞共孵育48h时，细胞内约有38％～46％的HBIG。浓度为0．1～10.0IU/mL的HBIG作用第3、6．9天时能抑制HepG2，2．15细胞培养上清中HBsAg、HBV DNA的分泌（P〈0．01）。住尤药物继续培养的第5、7天，培养卜清中HBsAg浓度较有药物培养的第3天低且继续下降（P〈0．01），第9～11天逐步增高；培养上清HBV DNA水平在第5天时也较有药物培养的第3天低并继续下降（P〈0．01），第7～11大时逐步增高。结论在体外细胞实验中，HBIG能跨膜转运入肝细胞内，细胞内外的HBIG能抑制HBsAg、HBV DNA的分泌。

Objective To investigate in vitro transmembrane transportation of hepatitis B immunoglobulin （HBIG） through hepatocytes and inhibition of HBsAg release and HBV replication in hepatocyte- derived cell line. Methods QSG7701 cells were cultured with DMEM containing different concentrations of HBIG. At different time points after culture, the supernatant was collected for HBsAb quantitative detection by time-resolved immunofluorometric assay （IFMA） and the amount of intraeellular HBIG was calculated. HepG 2. 2. 15 cells were cuhured with I）MEM in the absence or present of different concentrations of HBIG. The supernatant at some days interval was collected for HBsAg quantitative detection by IFMA and HBV DNA quantitative detection by real-time fluorescence quantitative PCR（RTFQ-PCR）. MTT assay was used to evaluate the cytotoxicity of HBIG. Results （1） 38%-46% of 0.1-0. 4 IU/mL HBIG was endocytosed into QSG7701 after 48 hours of culture. （2） HBIG in the concentration from 0. 01 IU/mL to 10. 0 IU/ml. had no cytotoxicity to the cells of QSG7701 and HepG2.2. 15. （3） HBsAg and HBV DNA in supernatant of HepG2.2. 15 cultured with O.1-10.0 IU/ml. HBIG reduced significantly than that of HepG2. 2. 15 cultured without HBIG after 3, 6, 9 days incubation, respectively （P〈0.01）. （4） The amount of HBsAg secreted in supernatant reduced continuously at the 5th and 7th day after HBIG was removed, （P〈O.01）, but rebounded at the 9th （P〈0.01） and 11th day after HBIG was removed, respectively, as compared with the control. The amount of HBV DNA secreted in supernatant reduced continuously at the 5th day after HBIG was removed （P〈0.01）, but rebounded at the 7th, 9th and 11 t h day after HBIG was removed, respectively, as compared with the control. Conclusion HBIG can be endocytosed into hepatocytes and extracellular or intracellular HBIG can inhibit the secretion of HBsAg and HBV DNA in vitro.