HBcAg肽特异性CD8^+T细胞抑制乙型肝炎病毒的体外实验研究

HBcAg-specific CD8^+T Cells Inhibit HBV Replication in vitro

目的观察HBV感染者的HBcAg肽特异性CD8+T细胞体外抑制乙型肝炎病毒(HBV)的作用和探讨非溶细胞机制清除病毒的效应分子。方法以HepG2.2.15细胞为靶细胞。用HLA-A匹配的HBcAg肽特异性CD8+T细胞克隆(效应细胞)与靶细胞(效靶比例1∶50)共同培养,于24h、48h和72h收集培养上清,通过检测其中HBV产物的变化,观察CD8+T克隆对HBV的抑制作用。用抗体中和法观察CD8+T细胞分泌的IFN-γ被封闭后HBV抑制的变化。结果 HBV特异性CD8+T克隆与靶细胞共育后,培养上清可检出高水平IFN-γ。共育后对HBsAg、HBeAg和HBV-DNA的最高抑制率分别为54.55%、50.36%和74.55%,均在72h。IFN-γ被抗体封闭后,对HBV DNA的抑制率显著下降,24h和48h分别为6.22%和17.48%。细胞毒活性最高见于24h,15.66%。结论①病毒特异性CD8+T细胞对靶细胞中HBV的清除既有溶细胞机制,也有非溶细胞机制参与;②IFN-γ是非溶细胞机制清除病毒的主要效应分子。

Objective To investigate the effects of HBcAg - specific CD8^ ＋ T cells on inhibiting HBV replication in vitro,and to search the cytokine of noncytolytic mechanisms in viral clearance. Methods By the method of coculture of HepG2.2. 15 cell （ target cells） with HLA - A2 matched HBcAg - specific CD8^ ＋ T cell clone （ effector cells） at E ： T ratios of 1 ： 50, and monitoring HBV production （HBsAg,HBeAg, and HBV- DNA）in coculture supernatants at 24h,48h and 72h, the percentage of decrease in HBV replication level was observed. Furthermore, blocking experiment with neutralizing mAbs to IFN -γ was performed to evaluate the effect of this cyto- kine. Results CD8 ^＋ T clone produced high levels of IFN - γ following coculture with 2.2.15 ceils. HBsAg,HBeAg and HBV - DNA in cocuhure supernatants were significantly reduced, and the greatest effect was observed at 72h by 54.55 % ,50.36% and 74.55% , respectively. The reduction of HBV DNA was decreased followed by using neutralizing mAbs to IFN - γ. The maximum activity of cytotoxicity of target ceils was at 24h by 15.66%. Conclusion ①HBV - specific CD8^＋ T cells inhibit HBV replication by cytolytic and noncytolytic mechanisms.②The effect of noncytolytic mechanisms is mainly mediated by IFN - γ.