入胞抑制剂Myrcludex-B合成肽对HBV感染HepaRG细胞体外感染模型的抑制作用研究

Study of the inhibitory effect of cell entry inhibitory myrcludex-B synthetic peptide on HepaRG cells infected with hepatitis B virus in an in vitro infection model

目的建立乙型肝炎病毒(HBV)阳性血清体外感染人肝癌细胞(HepaRG)的实验模型,并探索入胞抑制剂Myrcludex-B在体外感染模型中对HBV的抑制作用。方法将诱导成熟的HepaRG细胞分成加聚乙二醇(PEG)的感染组和无PEG的感染组,再将两种感染组分别分为添加Myrcludex-B肽段的处理组和不添加Myrcludex-B肽段的对照组,用HBV DNA阳性患者的血清对细胞进行感染。取细胞上清液,用荧光定量PCR检测上清液中HBV DNA的表达量,用化学荧光法检测细胞上清液中HBsAg、HBeAg的含量。两组间计量资料不服从正态分布的采用秩和检验,服从正态分布的采用t检验,以P<0.05为差异有统计学意义。结果在处理组中,加PEG感染组和不加PEG感染组的病毒滴度分别为(103877.00±49013.24)拷贝/ml、(5050.09±631.26)拷贝/ml;在对照组中,加PEG感染组和不加PEG感染组的病毒滴度分别为(116188.57±50957.19)拷贝/ml、(5119.34±1102.43)拷贝/ml,两组中加PEG感染组的病毒滴度显著高于不加PEG感染组(P<0.05),差异有统计学意义。在加PEG的感染组中,加肽段处理组和无肽段对照组的病毒滴度分别为(103877.34±49013.24)拷贝/ml、(116188.57±50957.19)拷贝/ml,加肽段处理组病毒滴度明显下降(P<0.05),差异有统计学意义;该组0、1、2、3 d上清液中HBsAg的含量均为阳性,且0 d上清液中加肽段处理组的HBsAg较无肽段对照组呈明显下降趋势。结论用添加PEG的HBV DNA阳性血清体外感染HepaRG细胞的实验模型是可行的,且在该试验模型中,入胞抑制剂Myrcludex-B对HBV感染存在一定的抑制作用。

Objective To establish and explore the inhibitory effect of cell entry inhibitor myrcludex-B on human liver cancer cell(HepaRG)infected with hepatitis B virus(HBV)positive serum in an in vitro infection model.Methods HepaRG induced mature cells were divided into polyethylene glycol(PEG)infection group and non-PEG infection group,and then the two infection groups were divided into treatment group with myrcludex-B peptide fragment and control group without myrcludex-B peptide fragment,and the cells were infected with the serum of HBV DNA-positive patients.The expression of HBV DNA in supernatant was detected by fluorescence quantitative PCR,and the HBsAg and HBeAg content in supernatant were detected by chemical fluorescence.Measurement data of the non-normal distribution between the two groups was determined by rank-sum test,and normal distribution was determined by t-test.P<0.05 was considered as statistically significant.Results In the treatment and control group the virus titers of the PEG infection group and the non-PEG infection group were(103877.00±49013.24)copies/ml and(5050.09±631.26)copies/ml,and(116188.57±50957.19)copies/ml,(5119.34±1102.43)copies/ml,respectively.Virus titers of both groups with PEG infection were significantly higher than non-PEG infection group,and the difference was statistically significant(P<0.05).In the PEG infection group,the virus titers of the peptide-treated group and the non-peptide control group were(103877.34±49013.24)copies/ml and(116188.57±50957.19)copies/ml,respectively,and the virus titers of the peptide-treated group was decreased significantly(P<0.05),and the difference was statistically significant.HBsAg content in the supernatant of this group was positive at 0,1,2 and 3 days,while the 0 day HBsAg supernatant treated with peptide fragment was significantly lower than that of the control group without peptide fragment.Conclusion It is feasible to infect HepaRG cells with HBV positive serum supplemented with PEG in vitro,and the cell entry inhibitor myrcludex-B has a certain inhibitory effect on hepatitis B virus infection in this experimental model.