CAJ | 학술논문

目的观察融合蛋白PTD-HBcAg诱导体外培养的小鼠髓源性树突状细胞(DC)成熟及对T淋巴细胞增殖的作用.方法体外分离培养近交系BALB/C小鼠髓源性DC加入重组粒细胞-巨噬细胞集落刺激因子(rgM-CSF)、重组IL-4培养5 d,再加人TNF-a、HBcAg和PTD-HBcAg诱导DC成熟.激光共聚焦显微镜观察免疫荧光在细胞中的分布及定位,流式细胞计数仪测定DC表面分子表达,ELISA法测定DC培养上清液中IL-12 p70的水平,CCK-8试剂盒检测T淋巴细胞增殖反应.组间数据比较采用t检验.结果成功体外诱导培养小鼠髓源性DC,HBcAg主要定位于DC膜表面,而PTD-HBcAg能够穿透DC膜进入细胞质.PTD-HBcAg能明显上调DC表面分子CD80、CD86和主要组织相容性复合体(MHC)II类分子表达;50 mg/L和100 mg/L PTD-HBcAg诱导DC分泌IL-12 p70水半分别为(142.50±18.31)ng/L和(124.30±15.12)ng/L,明显高于HBcAg诱导组的(42.31±4.21)ng/L(t=9.234和9.045,均P＜0.05);PTD-HBcAg诱导DC刺激T淋巴细胞增殖能力明显高于HBcAg组及阳性对照TNF-a组.结论 PTD-HBcAg具有穿透DC膜能力,并能促进DC分化、成熟,明显上调表面共刺激分子表达,增强DC刺激T淋巴细胞增殖能力及分泌IL-12 p70的水平.
목적 관찰융합단백PTD-HBcAg유도체외배양적소서수원성수돌상세포(DC)성숙급대T림파세포증식적작용.방법 체외분리배양근교계BALB/C소서수원성DC가입중조립세포-거서세포집락자격인자(rgM-CSF)、중조IL-4배양5 d,재가인TNF-a、HBcAg화PTD-HBcAg유도DC성숙.격광공취초현미경관찰면역형광재세포중적분포급정위,류식세포계수의측정DC표면분자표체,ELISA법측정DC배양상청액중IL-12 p70적수평,CCK-8시제합검측T림파세포증식반응.조간수거비교채용t검험.결과 성공체외유도배양소서수원성DC,HBcAg주요정위우DC막표면,이PTD-HBcAg능구천투DC막진입세포질.PTD-HBcAg능명현상조DC표면분자CD80、CD86화주요조직상용성복합체(MHC)II류분자표체;50 mg/L화100 mg/L PTD-HBcAg유도DC분비IL-12 p70수반분별위(142.50±18.31)ng/L화(124.30±15.12)ng/L,명현고우HBcAg유도조적(42.31±4.21)ng/L(t=9.234화9.045,균P＜0.05);PTD-HBcAg유도DC자격T림파세포증식능력명현고우HBcAg조급양성대조TNF-a조.결론 PTD-HBcAg구유천투DC막능력,병능촉진DC분화、성숙,명현상조표면공자격분자표체,증강DC자격T림파세포증식능력급분비IL-12 p70적수평.
Objective To observe the effects of PTD-hepatitis B core antigen (HBcAg) induced murine bone marrow-derived dendritic cells (DCs) maturation on T lymphocyte proliferation in vitro.Methods Bone marrow derived DCs isolated from BALB/c mice were cultured with recombinant granu|ocyte-macrophage colony-stimulating factor (rGM-CSF) and recombinant interleutin-4 (rIL-4)for 5 days.Tumor necrosis factor (TNF)-a,HBcAg and PTD-HBcAg were added to induce DCs maturation.The distribution and localization of intracellular immunofluorescence were observed by confocal microscopy,and DCs phenotypes were analyzed by flow cytometry.The level of IL-12 p70 in the supernatant was detected by enzyme linked immunosorhent assay (ELISA).The proliferation of T lymphocytes was performed by using cell counting kit-8 (CCK-8).All data were analyzed using t test.Results DCs were cultured and identified successfully.Recombinant PTD-HBcAg could penetrate into DCs cytoplasm while recombinant HBcAg was detected on the surface of cells.DCs surface molecules,such as CD80,CD86 and major histocompability complex (MHC) II were upregulated by PTDHBcAg;IL-12 p70 levels induced by 50 mg/L and 100 mg/L recombinant PTD-HBcAg were (142.50±18.31) ng/L and (124.30±15.12) ng/L,respectively,which were significantly higher than those induced by recombinant HBcAg [(42.31±4.21 ) ng/L,t = 9.234 and 9.045,respectively,P＜0.05].The proliferation of T lymphocytes induced by PTD-HBcAg was much higher than that in HBcAg group or positive control TNF-a group.Conclusions PTD-HBcAg could penetrate membrane of DCs and promote the differentiation and maturation of DCs.PTD-HBcAg could up-regulate the expressions of costimulatory molecules on cell surface of DCs,and enhance the ability of DCs on stimulating T lymphocytes proliferation and IL-12 p70 production.