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目的探讨重组人乙型肝炎病毒核心抗原(rhHBcAg)诱导髓源性树突状细胞(mDC)分泌白细胞介素15(IL-15)的机制。方法采用人单核细胞磁珠负分选试剂盒分选外周血单核细胞,采用粒细胞-巨噬细胞集落刺激因子(GM-CSF)、IL-4和肿瘤坏死因子α(TNF-α)诱导分化为mDC,10μg/m L rhHBcAg刺激mDC 1~6 d;分别用25μmol/L磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号通路抑制剂LY294002、1μmol/L p38丝裂原激活蛋白激酶(p38MAPK)信号通路抑制剂SB203580、100 nmol/L c-Jun N末端激酶(JNK)信号通路抑制剂SP600125和150 nmol/L胞外信号调节激酶(ERK)信号通路抑制剂U0126预处理1 h,再给予10μg/m L rhHBcAg刺激48 h。之后收集细胞和细胞培养上清,实时定量PCR检测IL-15 mRNA水平,ELISA检测IL-15蛋白水平,Western blot法检测PI3K/Akt和MAPK信号通路分子磷酸化水平。结果与rhHBcAg未刺激组比较,rhHBcAg刺激组IL-15 mRNA和蛋白水平显著升高;与rhHBcAg刺激组比较,p38抑制剂预处理组IL-15水平明显降低,但PI3K/Akt、JNK和ERK抑制剂预处理组IL-15水平未见显著性改变。结论 rhHBcAg通过p38MAPK信号通路上调外周血mDC IL-15的分泌。
Objective To investigate the mechanism of recombinant human Hepatitis B virus core antigen (rhHBcAg) inducing interleukin 15 (IL-15) secretion by myeloid dendritic cells (mDC). Methods Peripheral blood mononuclear cells (PBMCs) were sorted by human monocytes magnetic beads negative selection kit. GM-CSF, IL-4 and tumor necrosis factor-α (TNF- The cells were induced to differentiate into mDC and 10μg / ml rhHBcAg for 1 ~ 6 days. The cells were treated with 25μmol / L phosphatidylinositol 3 kinase / protein kinase B (PI3K / Akt) inhibitor LY294002 and 1μmol / L p38 mitogen Activation of p38MAPK signaling pathway inhibitor SB203580, 100 nmol / L c-Jun N-terminal kinase (JNK) signal pathway inhibitor SP600125 and 150 nmol / L extracellular signal-regulated kinase (ERK) signaling pathway inhibitor U0126 pretreatment 1 h, and then given 10μg / m L rhHBcAg stimulation 48 h. The cells and cell culture supernatants were collected. The levels of IL-15 mRNA were detected by real-time quantitative PCR. The levels of IL-15 protein were detected by ELISA. The phosphorylation of PI3K / Akt and MAPK signal pathway was detected by Western blot. Results Compared with rhHBcAg unstimulated group, IL-15 mRNA and protein levels were significantly increased in rhHBcAg-stimulated group. Compared with rhHBcAg-stimulated group, the level of IL-15 in p38 inhibitor pretreatment group was significantly decreased, but PI3K / Akt, JNK and ERK No significant change of IL-15 level was found in the pretreatment group. Conclusion rhHBcAg up-regulates the secretion of mDC IL-15 in peripheral blood by p38 MAPK signaling pathway.