论文部分内容阅读
目的:探讨葫芦茶苷体外抗乙型肝炎病毒的作用以及对JAK/STAT信号通路的调控作用机制。方法:以HBV基因转染的HepG2.2.15体外细胞模型,采用MTT法检测葫芦茶苷对HBV基因转染的HepG2.2.15细胞的半数毒性浓度(TC50)和最大无毒浓度(TC0),在最大无毒浓度(TC0)情况下观察不同浓度药物作用于HepG2.2.15细胞后,分别在第4 d和8 d收集细胞培养上清液,采用ELISA法测定上清液HBsAg,HBe Ag的滴度。FQ-PCR法检测上清液HBV DNA的含量;采用FQ-PCR法检测葫芦茶苷10μg/ml,20μg/ml,40μg/ml剂量组对HepG2.2.15细胞内信号转导和转录活化因子STAT1mRNA、STAT2mRNA表达的影响;RTPCR法检测JAK2、STAT3mRNA表达水平。结果:葫芦茶苷对HepG2.2.15细胞毒性较低,TC50为109.56μg/ml,TC0为41.54μg/ml。与空白对照组比较,10μg/ml,20μg/ml,40μg/ml剂量的葫芦茶苷能有效抑制HepG2.2.15细胞分泌HBsAg和HBe Ag,明显降低HBV DNA的含量。10μg/ml,20μg/ml,40μg/ml剂量的葫芦茶苷能明显升高细胞内信号转导和转录活化因子STAT1、STAT2、STAT3、JAK2 mRNA的水平。结论:葫芦茶苷体外具有显著的抗乙型肝炎病毒作用,其机制可能是通过激活JAK/STAT信号转导通路而发挥抗病毒作用。
Objective: To investigate the effect of gourdin on anti-hepatitis B virus (HBV) in vitro and the regulatory mechanism of JAK / STAT signal pathway. Methods: HepG2.2.15 cells in vitro transfected with HBV gene were used to detect the half-value concentration (TC50) and the maximum non-toxic concentration (TC0) of clotidine on HepG2.2.15 cells transfected with HBV gene. Nontoxic concentration (TC0) observed under different concentrations of drugs acting on HepG2.2.15 cells were collected on the 4th and 8th day after the cell culture supernatant was measured by ELISA supernatant HBsAg, HBe Ag titer. FQ-PCR method was used to detect the content of HBV DNA in supernatant. FQ-PCR was used to detect the expression of signal transducer and activator of transcription (STAT1) mRNA and protein in HepG2.2.15 cells by 10μg / ml, 20μg / ml and 40μg / STAT2mRNA expression; RTPCR method to detect JAK2, STAT3mRNA expression level. Results: Cucurbitacide had a lower cytotoxicity to HepG2.2.15 with TC50 of 109.56μg / ml and TC0 of 41.54μg / ml. Compared with the blank control group, the doses of gourdin at doses of 10μg / ml, 20μg / ml and 40μg / ml could effectively inhibit the secretion of HBsAg and HBe Ag from HepG2.2.15 cells and significantly reduce the content of HBV DNA. Hulukinin at doses of 10μg / ml, 20μg / ml and 40μg / ml significantly increased the levels of signal transducers and activators STAT1, STAT2, STAT3 and JAK2. Conclusion: Huluglucoside has a significant anti-HBV effect in vitro. The mechanism may be through antiviral action by activating JAK / STAT signal transduction pathway.