人参囊泡调控肿瘤免疫微环境的活性成分研究

目的 探讨人参Panx ginseng来源的细胞外囊泡（ginseng-derived nanoparticles，GDNPs）在调控肿瘤相关巨噬细胞表型及抑制黑色素瘤生长方面的潜在物质基础。方法 采用纳米颗粒跟踪分析仪、透射电镜测试及紫外-可见分光光度法全面表征GDNPs及其主要成分（蛋白质、多糖和皂苷）的含量；通过qRT-PCR和流式细胞术检测GDNPs与其含有的多糖、皂苷成分对骨髓来源巨噬细胞（bone marrow-derived macrophage，BMDM）表型的调控影响。收集不同极化程度的BMDM条件培养基（conditional medium，CM）孵育小鼠皮肤黑色素瘤B16F10细胞，CCK-8法测定不同CM处理后B16F10细胞活力，验证活性成分对肿瘤免疫微环境的调控作用；通过PMP柱前衍生法定量分析GDNPs活性成分组成。结果 透射电镜下观察GDNPs形态结构良好，含量测定结果为2.46×10¹¹颗粒的GDNPs含有4.31 mg蛋白质、4.46 mg多糖、1.22 mg皂苷。qRT-PCR和流式细胞术实验结果显示GDNPs多糖可以逆转M2型巨噬细胞表型，向M1方向极化。GDNPs多糖诱导巨噬细胞极化后的CM显著抑制了B16F10细胞活力。PMP柱前衍生法分析GDNPs多糖成分由葡萄糖、半乳糖、阿拉伯糖组成，其物质的量比为4.72∶1.07∶2.15。结论 揭示了GDNPs中多糖成分在调控肿瘤免疫微环境的关键作用，为进一步的机制研究和临床应用提供了实验依据。

Active components of ginseng vesicles regulating tumor immune microenvironment

Objective To explore the potential material basis of ginseng-derived nanoparticles (GDNPs) in regulating tumor-associated macrophage phenotype and inhibiting melanoma growth. Methods GDNPs and their major components (proteins, polysaccharides and saponins) were comprehensively characterized using nanoparticle tracking analyzer, transmission electron microscopy test and UV-Vis spectrophotometry. The regulatory effects of GDNPs and their polysaccharide and saponin components on the phenotype of bone marrow-derived macrophages (BMDM) were examined by qRT-PCR and flow cytometry experiments. B16F10 cells were incubated in BMDM conditioned medium (CM) with different degrees of polarization, and the viability of B16F10 cells was determined by CCK-8 assay to verify the modulation of the tumor immune microenvironment by the active ingredients. The active ingredient composition of GDNPs was quantified by PMP pre-column derivatization. Results The morphology and structure of GDNPs were well observed under transmission electron microscopy. 2.46 × 10¹¹ particles of GDNPs contained 4.31 mg of protein, 4.46 mg of polysaccharides, and 1.22 mg of saponins. qRT-PCR and flow cytometry experiments showed that the polysaccharide group of GDNPs reversed the phenotype of the M2-type macrophage cells, which were polarized toward M1. GDNPs polysaccharides induced macrophage polarization after CM significantly inhibited the viability of B16F10 cells. PMP pre-column derivatization analysis of the polysaccharide components of GDNPs consisted of glucose, galactose, arabinose, and the substance amount ratio was 4.72:1.07:2.15. Conclusion The experimental results reveal the key role of the polysaccharide components of GDNPs in the modulation of the tumor immune microenvironment, and provide the experimental basis for further mechanism study and clinical application.