PTD—HBcAg体外诱导小鼠髓源性树突状细胞成熟及在T淋巴细胞增殖中的作用

Effects of PTD-HBcAg induced murine bone marrow-derived dendritic cells maturation on T lymphocyte proliferation in vitro

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，加入重组粒细胞一巨噬细胞集落刺激因子（rGM-CSF）、重组IL4培养5d，再加入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-12p70的水平，CCK8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；50mg／L和100mg／LPTD-HBcAg诱导DC分泌IL-12p70水平分别为（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〈O．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-α组。结论PTD-HBcAg具有穿透IX；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Ib-2p70的水平。

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 granulocyte-macrophage colony-stimulating factor （rGM-CSF） and recombinant interleutin-4 （rlL-4） for 5 days. Tumor necrosis factor （TNF）-α, HBcAg and PTD-HBcAg were added to induce DCs maturation. The distribution and localization of intracellular immunofluorescence were observed by confocal microscopy, and Des phenotypes were analyzed by flow cytometry. The level of IL-12 pT0 in the supernatant was detected by enzyme linked immunosorbent 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 PTD- HBeAg; 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-α 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.