目的 构建人参多糖-壳寡糖纳米粒（Ginseng polysaccharide-chitosan oligosaccharide nanoparticles，GP-NPs），并探究其联合放疗能否通过激活I型干扰素（interferon-I，IFN-I）介导的Janus激酶3（Janus kinase 3，JAK3）/信号转导和转录激活蛋白5（signal transducer and activator of transcription 5，STAT5）信号通路，促进自然杀伤（natural killer，NK）细胞向高细胞毒性亚群分化，从而增强对非小细胞肺癌的抗肿瘤免疫应答。方法 通过离子交联法制备GP-NPs，并对其粒径、分散性及稳定性进行表征。建立A549肺癌细胞皮下荷瘤裸鼠模型，将小鼠分为PBS组、单纯放疗组和GP-NPs联合放疗组。通过测量肿瘤体积、ELISA检测血清细胞因子水平、流式细胞术分析NK细胞亚群、Western blotting检测JAK3/STAT5通路蛋白表达，以及组织病理学分析，综合评价GP-NPs联合放疗的抗肿瘤效果与机制。结果 GP-NPs联合放疗显著抑制肿瘤生长，且效果显著优于单纯放疗组（P＜0.01）。联合治疗组血清中γ干扰素（interferon-γ，IFN-γ）、白细胞介素-15（interleukin-15，IL-15）、颗粒酶和穿孔素水平显著升高（P＜0.01）。流式细胞术显示肿瘤内NK细胞浸润比例增加（P＜0.01），且成熟细胞毒亚群（CD27^-CD11b⁺）比例显著上升（P＜0.01）。Western blotting结果表明联合治疗显著激活JAK3/STAT5信号通路（P＜0.01）。组织学分析显示联合治疗组肿瘤细胞凋亡增加、DNA损伤加重、NK细胞浸润增强，且主要脏器未见明显毒性。结论 GP-NPs联合放疗通过激活IFN-I介导的JAK3/STAT5信号通路，促进NK细胞向高细胞毒性亚群分化，增强其抗肿瘤功能。该联合策略不仅显著提升放疗的疗效，还具有良好的生物安全性，为非小细胞肺癌的免疫联合治疗提供了新的实验依据和策略方向。

Objective To construct Ginseng polysaccharide-chitosan oligosaccharide nanoparticles (GP-NPs) and investigate whether their combination with radiotherapy could promote the differentiation of natural killer (NK) cells into highly cytotoxic subsets by activating interferon-I (IFN-I)-mediated Janus kinase 3 (JAK3)/signal transducer and activator of transcription 5 (STAT5) signaling pathway, thereby enhancing the antitumor immune response against non-small cell lung cancer (NSCLC). Methods GP-NPs were prepared via the ionic cross-linking method, and their particle size, dispersibility and stability were characterized. A subcutaneous xenograft model of A549 lung cancer cells in nude mice was established, and the mice were divided into PBS group, radiotherapy alone group and GP-NPs combined with radiotherapy group. The antitumor efficacy and mechanism of GP-NPs combined with radiotherapy were comprehensively evaluated by measuring tumor volume, detecting levels of cytokines in serum via ELISA, analyzing NK cells subsets by flow cytometry, detecting the protein expressions of JAK3/STAT5 pathway through Western blotting and performing histopathological analysis. Results GP-NPs combined with radiotherapy significantly inhibited tumor growth, and the effect was significantly better than that of the simple radiotherapy group (P < 0.01). The levels of interferon-γ (IFN-γ), interleukin-15 (IL-15), granzyme and perforin in serum of combined treatment group were significantly increased (P < 0.01). Flow cytometry showed an increase in the proportion of NK cells infiltration within the tumor (P < 0.01), and a significant increase in the proportion of mature cytotoxic subgroups (CD27^-CD11b⁺) (P < 0.01). Western blotting results showed that the combination therapy significantly activated the JAK3/STAT5 signaling pathway (P < 0.01). Organizational analysis showed that the combination therapy group had increased tumor cell apoptosis, aggravated DNA damage and enhanced NK cell infiltration, with no significant toxicity observed in major organs. Conclusion GP-NPs combined with radiotherapy promote the differentiation of NK cells into highly cytotoxic subsets and enhance their antitumor function by activating IFN-I-mediated JAK3/STAT5 signaling pathway. This combination strategy not only significantly improves the efficacy of radiotherapy but also has good biosafety, providing new experimental evidence and strategic directions for the immunocombination therapy of NSCLC.

R285.5

北京市科技新星计划（20240484544）;中国原子能科学研究院稳定性支持经费科研项目（CNNCWZ-2023002）