猪苓多糖经TLR4刺激小鼠骨髓来源的树突状细胞成熟

Polyporus polysaccharides regulate the phenotypic and functional maturation of murine dendritic cells

目的:研究猪苓多糖(polyporus polysaccharides,PPS)诱导小鼠骨髓树突状细胞(dendriticcell,DC)表型及功能成熟的分子机制。方法:从小鼠骨髓中分离单个核细胞(MNC),用rmGM-CSF、IL-4培养5 d后将其分为三组:实验组中加入50μg/mL PPS;阳性对照组中加1μg/mL LPS;加等量RPMI 1640培养基作为阴性对照组,继续培养48 h。苏木精-伊红(HE)染色观察细胞形态;流式细胞仪(FACS)分析DC表面CD80、CD86及MHC II(I-A/I-E)表达情况;经20μg/mL Toll-like receptor(TLR)4、TLR2单抗作用1 h后,ELISA法检测培养上清中IL-12 p40含量;混合淋巴细胞反应(MLR)检测DC对T淋巴细胞的刺激能力。结果:PPS处理的DC具有毛刺状突起,呈典型的DC形态;细胞表达MHC II、CD80及CD86增加;对T淋巴细胞刺激能力增强;分泌IL-12增加且可被抗TLR4单抗所阻断。结论:PPS通过TLR4促进体外培养的小鼠骨髓DC表型与功能成熟。

Objective：To study the molecular mechanisms by which polyporus polysaccharides（PPS） induce phenotypic and functional changes in human dendritic cells（DCs） in vitro,and stimulate the maturation of DC.Methods：Mononuclear cells（MNCs） isolated from murine bone marrow were cultured in RPMI 1640 medium containing rmGM-CSF and IL-4 for 5 days,and were then divided into three groups.The MNCs were stimulated for further 48 h with either 50 mg/L PPS,1 mg/L LPS（positive control） or RPMI 1640（negative control）.Morphological changes of the DCs were identified using hematoxylin/eosin staining.The expression of CD80,CD86,MHC II and I-A/I-E on DCs was analyzed by FACS.After simulation for 1 h with 20μg/mL Toll-like receptor（TLR） 4,TLR2 and the level of IL-12 p40 in the culture supernatant were detected by ELISA.The ability of PPS-induced DC to stimulate T cell maturation was determined by the 3H-TdR incorporation method.Results：PPS-treated DCs displayed a more mature morphology,with long protrusions.PPS increased the expression of I-A/I-E,CD80 and CD86 on the DC surface.PPS-treated DCs secreted higher levels of IL-12 p40 than that of untreated DCs.Neutralization with antibodies against TLR4 inhibited PPS-induced production of IL-12 p40.Conclusion：PPS stimulates the phenotypic and functional maturation of DCs derived from peripheral bone marrow monocytes via TLR4.