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目的了解双歧杆菌基因组DNA(bDNA)的免疫调节作用、双歧杆菌制剂抗过敏可能的机制。方法使用bDNA体外刺激脐带血单个核细胞(CBMC),以空白、DNaseⅠ完全酶解的bDNA(d-bDNA)和人DNA(hDNA)作为对照。在刺激后不同时间点终止培养,测定培养上清中细胞因子IL-12、IL-10的水平;计数分泌IFN-γ和IL-4的阳性细胞数量;并检测细胞内信号蛋白T-bet(T-box expressed in T cells)、GATA3 mRNA表达强度的变化。结果刺激12 h bDNA组细胞培养上清IL-12水平明显高于其他对照组(P<0.05),这种IL-12水平升高也发生在刺激后24、48 h。刺激12、24 h后,bDNA组细胞上清中IL-10水平比对照组、d-bDNA组和hDNA有所降低,但无统计学意义(P>0.05)。bDNA组加佛波脂(PMA)刺激后,分泌IFN-γ的阳性细胞数高于对照组(P<0.05)。bDNA组分泌IL-4阳性细胞数少于对照组、d-bDNA组及hDNA组(P<0.05)。bDNA组细胞在6、12、24 h后,细胞中核转录因子T-betmRNA的表达强度比对照组增强(P<0.05)。bDNA刺激不同时间后,细胞中核转录因子GATA3 mRNA的表达强度与其他组相比无明显差异。结论双歧杆菌基因组DNA上调CBMC IL-12、IFN-γ分泌及T-bet表达,下调IL-4分泌,促进CBMC Th1应答。这可能是双歧杆菌产生抗过敏作用的重要机制之一。
Objective To understand the immunomodulatory effect of Bifidobacterium genomic DNA (bDNA) and the possible anti-allergic mechanism of Bifidobacterium. Methods Human umbilical cord blood mononuclear cells (CBMCs) were stimulated with bDNA in vitro, and blank and DNase Ⅰ digested bDNA and human DNA (hDNA) were used as controls. At different time points after stimulation, the culture was terminated and the levels of cytokines IL-12 and IL-10 in the culture supernatant were measured. The number of positive cells secreting IFN-γ and IL-4 was counted. The expression of T-bet T-box expressed in T cells), GATA3 mRNA expression intensity changes. Results The level of IL-12 in 12 h bDNA group was significantly higher than that in other control groups (P <0.05). The increase of IL-12 level also occurred 24 and 48 h after stimulation. After stimulation for 12 and 24 h, the level of IL-10 in the bDNA group was lower than that in the control group, d-bDNA group and hDNA but not statistically significant (P> 0.05). The number of IFN-γ positive cells in bDNA group stimulated by PMA was higher than that in control group (P <0.05). The number of IL-4 positive cells in bDNA group was less than that in control group, d-bDNA group and hDNA group (P <0.05). The expression level of T-bet mRNA in the bDNA group was higher than that in the control group at 6, 12 and 24 h (P <0.05). After bDNA stimulation for different time, the expression intensity of GATA3 mRNA in the cells had no significant difference compared with other groups. Conclusion Bifidobacterium genomic DNA up-regulates CBMC IL-12, IFN-γ secretion and T-bet expression, down-regulates IL-4 secretion and promotes CBMC Th1 response. This may be one of the important mechanisms by which bifidobacteria produce anti-allergic effects.