双歧杆菌完整肽聚糖对脐血来源树突状细胞分化成熟的影响

Effect of whole peptidoglycan of Bifidobacterium on dendritic cell from cord blood

目的研究两歧双歧杆菌完整肽聚糖(WPG)对脐血来源树突状细胞(DC)形态及分泌细胞因子的影响,了解双歧杆菌WPG对DC分化、成熟及免疫调节功能的作用;并为益生菌及其生物活性成分的进一步开发提供依据。方法分离正常孕妇脐血单个核细胞诱导生成未成熟树突状细胞(Dendritic cells,DCs),实验组在培养的第7天分别加入两歧双歧杆菌WPG(5μg/ml)、两歧双歧杆菌全菌(100μg/ml),阳性对照组加入脂多糖(LPS),阴性对照组仅加入培养基。倒置显微镜在培养各期形态学观察,流式细胞术检测表面标志物CD83及CD1a的表达,MLR检测DCs刺激同种异体T淋巴细胞能力,ELISA法测定DCs培养上清中IL-12p70、IL-10的分泌。结果脐血单核细胞在双歧杆菌WPG与GM-CSF、IL-4协同诱导作用下,能成为形态上具有典型树突状突起的DCs;诱导后的CB-MDDCs刺激同种异体T细胞的增殖能力及分泌IL-12p70、IL-10的水平显著高于阴性对照组(P<0.01)且细胞表面标志物CD83及CD1a的表达增加。结论(1)双歧杆菌WPG能影响CB-MDDCs的成熟状态。(2)双歧杆菌WPG对CB-MDDCs成熟程度及分泌细胞因子水平的影响强于双歧杆菌全菌,说明WPG是双歧杆菌主要免疫活性成分。

Objective To explore in vitro the effect of the whole peptidoglycan （WPG） of Bifidobacterium on proliferation, maturation and immunoregulation of DCs from cord blood monocyte, and provide further evidence for the exploitation of probioties and its active ingredients. Method Cord blood monocular cells （CBMC） of normal expectant mother was isolated, cultured and induced to get immature DCs with GM-CSF and IL-4. Bifidobacterium bifidum and WPG of Bifidobacterium bifidum （5 μg/ml） was added into the experimental groups, respectively; Lipopolysaceharide （LPS） was added into the positive control group ; culture medium was added into the negative control group. The change of the shape of DCs was observed continuously with phase-contrast microscope and electron microscope. The expression of CD83 and CDla of DCs was detected with flow Cytometry （FC）. The capability of DCs to stimulate the proliferation of allogeneic T lymphoeytes was examined with MTr assay. The content of IL-12p70 and IL-10 in the culture supematant of DCs with enzyme linked immunosorbent assay （ELISA）. Result After induced with GM-CSF and IL-4, DCs from CBMC stimulated by the WPG of Bifidobacterium had typical morphology of mature DCs with enation. MLR test showed that the capability of the induced DCs to stimulate the proliferation of allogeneic T lymphocytes and secret IL-12p70 and IL-10 was more competent than that of the negative group （ P 〈0.01 ） ,and its expression of CD83 and CDla was higher than other groups. Conclusion （ 1 ） WPG has an effect on maturation of DCs from CBMCs. （ 2 ） WPG is more effective than Bifidobacterium in affecting the maturation degree of CB-MDDCs and eytokines production of CB-MDDCs, which suggests that WPG is one of the main active ingredients of Bifidobacterium.