双歧杆菌DNA上调脐带血单个核细胞Th1应答

Bifidobacterium DNA upregulates Th1 type response of umbilical cord blood mononuclear cell

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 study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell （CBMC）, and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction. Methods Bifidobactefium genomic DNA（bDNA） and human DNA（hDNA） were extracted with phenol/chloroform/isoamyl alcohol and stored at - 20℃ for later use. Parts of bDNA were completely digested with DNaseI （d-bDNA） at 37℃. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37℃ in a 5% CO2 humidified incubator. These cells were divided into four groups, control group： without any stimulant; bDNA group： stimulated with 25 μg/ml bDNA; d-bDNA group： stimulated with 25 μg/ml d-bDNA; hDNA group： stimulated with 25 μml hDNA. The cells were stimulated with different stimulants in vitro, at the end of incubation culture supernatant and cells were collected. IL-12 and IL-10 levels in the culture supernatant were measured by enzyme linked immuno sorbent assay （ELISA） ; cells secreting IL-4 and IFN-gamma were counted by enzyme linked immunospot （ELISPOT） assay; and total RNA was isolated from the cells to assay T-bet and GATA3 mRNA expression levels by reverse transcription polymerase chain reaction （RT-PCR）. Results Six hours after stimulation there was no significant difference in IL-12 level in supernatant among the four groups; 12 hours after stimulation, IL-12 level in supernatant of bDNA treated group was significantly higher than that of each of the other groups, so were the results obtained at 24 hours and 48 hours after stimulation （ P 〈 0. 05 ）. No significant difference could be detected in IL-12 level in supernatant among the other 3 groups. On the other hand, 6 hours after stimulation there was no significant difference in IL-10 level in supernatant among the four groups. But 12 and 24 hours after stimulation IL-10 level in supernatant of bDNA treated group was lower than that of each of the other groups, but the difference was not statistically significant. The count of IFN-γ secreting cells of bDNA treated group was higher than that of the other groups, while IL-4 secteting cells of bDNA treated group were lower than that of the other groups. After bDNA stimulation, nuclear factor T-box expressed in T cells （T-bot） mRNA expression was conspicuously enhanced as compared to the other three groups （ P 〈 0. 05 ）. GATA3 mRNA transcription in CBMC had no significant change after bDNA stimulation. Conclusion bDNA could promote secretion of Thl type cytokine IL-12, while Th2 type cytokine IL-10 level of cell supematant was decreased, bDNA could stimulate secretion of IFN-γ by CBMC and inhibit secretion of IL-4. T-bet mRNA expression was highly enhanced after bDNA stimulation, bDNA could upregulate Thl type response, which may be one of important mechanisms by which bifidobacterium inhibit allergic response.