【摘要】 目的:分离纯化猪苓多糖,观察猪苓多糖对人巨噬细胞的形态和免疫功能的影响,并进行机制分析。方法:甲醇脱脂、水提醇沉提取猪苓粗多糖,经过滤、Savage法去蛋白、冷冻干燥、DEAE-Cellulose及Sephadex G-100柱层析分离猪苓均一多糖(PPS);Ficoll法分离新生儿脐带血PBMC,并通过CD14磁珠分选及M-CSF诱导人M2型巨噬细胞;观察猪苓多糖对M2形态的影响;FACS检测细胞表型(CD14、CD68、CD86及CD206)、主要组织相容性复合体2(MHCⅡ)与程序性细胞死亡蛋白1(PD1)表达量;ELISA分析细胞因子[白细胞介素(IL)-1β、IL-2、IL-6、IL-8、IL-10、肿瘤坏死因子-α(TNF-α)、干扰素γ(IFN-γ)、转化生长因子-β(TGF-β)及重组人巨噬细胞集落刺激因子(GM-CSF)]含量;检测猪苓多糖对M2和肿瘤细胞共培养的杀伤能力;Western bolt分析TLR2、TLR4及NF-κB蛋白表达;RT-PCR测定层黏连蛋白(LN)、纤维黏连蛋白(FN)、Ⅲ型胶原蛋白(COLⅢ)、细胞间黏附分子-1(ICAM-1)和巨噬细胞表面分子抗原(MAC-1)m RNA水平。结果:分离纯化得到PPS;PPS影响巨噬细胞的黏附和诱变细胞形态;极化M1表型,降低PD1表达(P<0.01),升高MHCⅡ分子表达(P<0.05,P<0.01);促进细胞因子IL-1β、IL-6、IL-8、IL-10及TNF-α分泌(P<0.01);增强M2巨噬细胞对肿瘤杀伤的能力(P<0.05);升高TLR2、TLR4和NF-κB蛋白的表达(P<0.05,P<0.01);降低FN、COLⅢ和ICMA-1转录水平(P<0.05,P<0.01)。结论:PPS抑制M2巨噬细胞的黏附和伪足生成,且具有显著的免疫增强作用,其机制与激活TLR2/TLR4-NF-κB信号通路、下调黏附蛋白和因子有关。更多还原

【Abstract】 Objective: To observe the effects of Polyporus polysaccharides on morphological changes and immune function in human macrophages in vitro after isolating polysaccharides from Polyporus umbellaru(Pers.) Fr., and to analyze the mechanism. Methods: Polyporus polysaccharides were obtained by water extraction and alcohol precipitation, and through Savage method to remove the protein, freezing and drying, DEAE-Cellulose ion exchange chromatography and Sephadex G-100 filtration chromatography, uniform Polyporus polysaccharides were obtained. Ficoll method was used to isolate neonatal umbilical cord blood PBMC, through the CD14 magnetic beads for separation, and M2 macrophages were induced by M-CSF. The effects of Polyporus polysaccharides on the morphology of M2 was observed. Phenotypes(CD14, CD68, CD86 and CD206) and expressions of MHC I and PD1 in macrophages were detected by FACS. Levels of IL-1β, IL-2, IL-6, IL-8, IL-10, TNF-α, IFN-γ, TGF-α and GM-CSF were analyzed by ELISA. The killing effect of Polyporus polysaccharides on M2 co-culture of cancer cells was detected. Western bolt was used to detect protein expression of TLR2, TLR4 and NF-κB, and RT-PCR was used to detect m RNA expression of laminin(LN), fibronectin(FN), collagen I I(COLI I) and intercellular adhesion molecule-1(ICAM-1) and macrophage surface antigen(MAC-1). Results: After isolating and purifying, a uniform polysaccharide form Polyporus polysaccharides named PPS was obtained, which affected the adhesion and morphology changes in macrophage. It could polarize M1 phenotype, decrease the expression of PD1(P<0.01) and increase expression of MHC I(P<0.05, P<0.01). It promoted the secretion of cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α(P<0.01). It enhanced the tumor killing effects M2 macrophages(P<0.05), as well as it increased the protein expression of TLR2, TLR4 and NF-κB(P<0.05, P<0.01), but reduced the transcription of FN, COL I I and ICMA-1(P<0.05, P<0.01). Conclusion: PPS inhibites macrophage adhesion and pseudopod formation with a significant immunopotentiation, and its mechanism may be related to activation of TLR2/TLR4-NF-κB signaling pathway and down-regulation of adhesion proteins.更多还原