重组卡介苗rBCGAg85AESAT6对人巨噬细胞免疫刺激活性的影响

Effect of rBCGAg85AESAT6 on immunostimulatory activity of human macrophages

摘要:

目的探究重组卡介苗(rBCGAg85AESAT6,rBCGAE)对人巨噬细胞免疫刺激活性的影响，为该疫苗的应用提供重要的理论依据和实验基础。方法将rBCGAE（rBCGAE感染组）及卡介苗（BCG感染组）分别感染体外培养的受PMA诱导分化的人单核细胞系（THP1细胞株），分别于感染后24、48及72 h，观察细胞表面分子CD80和CD86的表达及细胞培养基中干扰素（IFN）γ及肿瘤坏死因子（TNF）ａ的诱生量。结果THP1细胞（未受PMA诱导分化的细胞）和PMA分化的THP1细胞培养4 h后，细菌吞噬率分别为（8.45±1.54）%、（91.26±2.13）%，后者显著高于前者（P=0.01）；感染后24、48及72 h，rBCGAE组CD86阳性细胞比率分别为 (32.84±7.13）%、（48.42±5.46）%、（39.48±5.67）%，CD80阳性细胞比率分别为(20.28±1.13）%、（23.67±1.23）%、（23.19±1.58）%，均显著高于BCG感染组的CD86［分别为（28.17±5.26）%、（40.09±7.21）%、（31.26±6.85）%］和CD80阳性细胞比率［分别为（22.15±1.82）%、（23.27±1.91）%、（22.68±0.87）%］，差异均有统计学意义（均P＜0.01)。感染后24、48及72 h，rBCGAE组IFNγ诱生量分别为(1 986±156）pg/mL、（4 687±168）pg/mL、（3 238±97）pg/mL，TNFа诱生量分别为(1 153±48）pg/mL、（5 864±97）pg/mL、（4 129±68)pg/mL，各时间点均显著高于BCG感染组的IFNγ［分别为（1 245±32）pg/mL、（3 067±143）pg/mL、（2 879±186）pg/mL］和 TNFа诱生量［分别为（486±18）pg/mL、（3 237±86）pg/mL、（1 068±74）pg/mL］，差异均有统计学意义（均P＜0.01)。结论rBCGAE可显著增强人巨噬细胞免疫刺激活性，该疫苗可作为替代BCG的候选疫苗，具有进一步开发研究的价值。

Abstract:

ObjectiveTo evaluate  the effect of recombinant bacillus CalmetteGuerin (BCG)rBCGAg85AESAT6（rBCGAE)on immunostimulatory activity of human macrophages, and  provide theoretical proof and experimental basis for  vaccine use.MethodsThe previously constructed rBCGAE and BCG strains were obtained respectively to infect the THP1 cells that stimulated by phorbol12myristate13acetate（PMA）. 24 h,48 h and 72 h after infection, expression of macrophage surface markers CD80 and CD86  were measured by flow cytometry, and the concentration of interferonγ (IFNγ) and tumor necrosis factorａ(TNFａ) in supernatant were detected with ELISA kit. ResultsAfter four hours culture, phagocytic rate of THP1  stimulated by PMA was  significantly higher than that nonstimulated by PMA （［91.26±2.13］% vs ［8.45±1.54］%，P=0.01）； 24 h,48 h and 72 h after infection, the percentages of CD86 positive cells (［32.84±7.13］%, ［48.42±5.46］%, ［39.48±5.67］%) and CD80 positive cells (［20.28±1.13%］, ［23.67±1.23］%, ［23.19±1.58］%) in rBCGAE group were obviously higher than those in BCG group (CD86：［28.17±5.26］%, ［40.09±7.21］%, ［31.26±6.85］%; CD80：［22.15±1.82］%, ［23.27±1.91］%, ［22.68±0.87］%, P<0.01). 24 h,48 h and 72 h after infection, the concentration of IFNγ (［1 986±156］pg/mL,［ 4 687±168］ pg/mL, ［3 238±97］ pg/mL) and TNFа(［1 153±48］ pg/mL, ［5 864±97］ pg/mL, ［4 129±68］pg/mL) in rBCGAE group were higher than those in BCG group (IFNγ：［1 245±32］pg/mL, ［3 067±143］ pg/mL, ［2 879±186］ pg/mL; TNFа：［486±18］ pg/mL, ［3 237±86］ pg/mL, ［1 068±74］ pg/mL, P<0.01).ConclusionRecombinant BCGAE can enhance immunostimulatory activity of human macrophages, and it is an improved TB vaccine as an alternative to BCG for further study.