CAJ | 학술논문

目的：探讨转乙型肝炎病毒（HBV）基因的小鼠树突状细胞（DCs）刺激自体淋巴细胞在体外对HBV复制的影响。方法采用转HBV基因的小鼠DCs，在体外用HBV表面抗原（HBsAg）和核心抗原（HBcAg）诱导DCs成熟。将成熟DCs与小鼠自体来源的淋巴细胞共刺激后形成特异性的免疫效应细胞（IEC），再与人肝癌细胞系HepG 2.2.15细胞共培养，采用生物化学、流式细胞仪、荧光实时定量聚合酶链反应（RT-PCR）和酶联免疫吸附试验（ELISA）等方法检测肝功能、HBV DNA、共价闭合环状DNA（cccDNA）水平和炎症相关因子的变化。结果诱导成熟的DCs与IEC共同作用HepG 2.2.15后，其细胞的形态、酶学无明显变化，但上清液中HBV DNA分泌明显减少，以及细胞内HBV DNA和cccDNA水平也明显降低，与未成熟DCs组比较差异有统计学意义。细胞上清液中炎症相关因子的分泌有明显变化：HBV DNA高表达时，IFN-γ和IL-2水平下降，而IL-10升高；HBV DNA低表达时，干扰素-γ（IFN-γ）和细胞白介素-2（IL-2）水平上升，而IL-10水平下降。结论 HBV相关抗原刺激成熟的转HBV小鼠的DCs与自体淋巴细胞共刺激后形成的特异性IEC，在体外对HBV复制具有抑制作用，细胞因子参与其变化。
목적：탐토전을형간염병독（HBV）기인적소서수돌상세포（DCs）자격자체림파세포재체외대HBV복제적영향。방법채용전HBV기인적소서DCs，재체외용HBV표면항원（HBsAg）화핵심항원（HBcAg）유도DCs성숙。장성숙DCs여소서자체래원적림파세포공자격후형성특이성적면역효응세포（IEC），재여인간암세포계HepG 2.2.15세포공배양，채용생물화학、류식세포의、형광실시정량취합매련반응（RT-PCR）화매련면역흡부시험（ELISA）등방법검측간공능、HBV DNA、공개폐합배상DNA（cccDNA）수평화염증상관인자적변화。결과유도성숙적DCs여IEC공동작용HepG 2.2.15후，기세포적형태、매학무명현변화，단상청액중HBV DNA분비명현감소，이급세포내HBV DNA화cccDNA수평야명현강저，여미성숙DCs조비교차이유통계학의의。세포상청액중염증상관인자적분비유명현변화：HBV DNA고표체시，IFN-γ화IL-2수평하강，이IL-10승고；HBV DNA저표체시，간우소-γ（IFN-γ）화세포백개소-2（IL-2）수평상승，이IL-10수평하강。결론 HBV상관항원자격성숙적전HBV소서적DCs여자체림파세포공자격후형성적특이성IEC，재체외대HBV복제구유억제작용，세포인자삼여기변화。
Objective To explore the effects of dendritic cells (DCs) from hepatitis B virus (HBV) transgenic mice-stimulated autologous lymphocytes on HBV replication in vitro. Methods DCs from HBV transgenic mice were induced to maturity by incubation with hepatitis B surface antigen(HBsAg)and hepatitis B core antigen (HBcAg) in vitro. Mature DCs and autologous lymphocytes were co-stimulated to form specific sensitized immune effector cells (IEC), then co-cultured with the human hepatoma cell line HepG 2.2.15. Changes in morphology and activity of hepatocytes were then observed,and liver enzyme as well as inflammatory cytokine levels in the culture supernatant were detected using enzyme linked immunosorbent assay (ELISA). Intracellular HBV DNA and covalently closed circular DNA (cccDNA) concentration were measured by real-time PCR. Results Co-stimulation by mature DCs and IEC showed no impact on the morphology and liver enzyme expression level of HepG 2.2.15 cells, but the supernatant HBVDNA and intracellular HBV DNA and cccDNA levels decreased significantly compared with those cells co-cultured with immature DCs. Secretion of inflammatory cytokines in the supernatant showed that when HBV DNA was highly expressed,the concentration of IFN-γand IL-2 decreased,while IL-10 was increased. Contrastingly, when HBV DNA had low expression,the concentration of IFN-γ and IL-2 increased,however,the expression of IL-10 decreased. Conclusion Co-stimulation of HBV-related antigen-induced mature DCs and autologous lymphocytes showed inhibitory effects on ex vivo HBV replication,and cytokines were suggested to mediate this effect.