[摘 要] 目的：探讨Ⅱ型固有淋巴细胞（ILC2）-双调蛋白（AREG）-调节性T（Treg）细胞轴在宫颈癌免疫微环境调控中的作用及机制。方法：收集2021年5月—2022年5月于新疆医科大学第一附属医院收治的Ⅰ-ⅡA期宫颈癌患者肿瘤组织（n = 8），以子宫肌瘤手术患者的正常宫颈组织（n = 8）作为对照；另收集全分期宫颈癌患者的外周血样本（n = 30），并以健康志愿者外周血作为对照（n = 30）。利用GEPIA数据库分析AREG与叉头框蛋白P3（Foxp3）的mRNA表达水平。采用多重免疫荧光技术、流式细胞术检测组织及外周血中ILC2、Treg细胞浸润水平；通过ELISA、IHC和WB法验证AREG、Foxp3及IL-10的表达水平，并对ILC2、AREG、Treg细胞及IL-10进行相关性分析。体外分离宫颈癌患者ILC2与PBMC，分别使用重组人IL-33（rhIL-33）、抗人IL-33抗体（α-IL-33）及重组人AREG（rhAREG）、抗人AREG抗体（α-AREG）进行干预；采用CCK-8法与流式细胞术检测不同浓度rhAREG对HeLa、SiHa细胞增殖及凋亡的影响，ELISA检测上清中AREG、IL-10浓度，流式细胞术检测Treg细胞比例变化。此外，本研究还比较了宫颈癌患者手术前后外周血中ILC2、AREG、Treg细胞及IL-10的水平差异。结果：宫颈癌患者组织及外周血中ILC2浸润水平和AREG表达显著高于对照组，Treg细胞比例、Foxp3及IL-10表达亦明显上调（P < 0.05）；相关性分析显示，ILC2、Treg细胞、AREG与IL-10彼此正向关联。体外实验表明，不同浓度rhAREG对宫颈癌细胞（HeLa、SiHa）的增殖及凋亡无明显作用（P > 0.05）；rhIL-33可激活ILC2并上调AREG分泌（P < 0.01），而α-IL-33可逆转该效应（P < 0.05）；rhAREG可促进Treg细胞分化及IL-10分泌（P < 0.001），α-AREG则显著逆转上述作用（P < 0.01）。此外，宫颈癌患者术后外周血中ILC2、Treg细胞、AREG及IL-10水平均显著降低（P < 0.0001）。结论：ILC2-AREG-Treg免疫调控轴异常激活，可能通过介导免疫抑制性肿瘤微环境形成参与宫颈癌进展。

[Abstract] Objective: To investigate the role and mechanism of the group II innate lymphoid cell (ILC2)-amphiregulin (AREG)-regulatory T (Treg) cell axis in the regulation of the immune microenvironment in cervical cancer. Methods: Tumor tissue samples were obtained from patients with stage I-IIA cervical cancer (n = 8) who were admitted to the First Affiliated Hospital of Xinjiang Medical University between May 2021 and May 2022. Normal cervical tissues from patients undergoing surgery for uterine fibroids served as controls (n = 8). Additionally, peripheral blood samples were collected from patients with cervical cancer at all stages (n = 30), with peripheral blood from healthy individuals serving as controls (n = 30). The mRNA expression levels of AREG and forkhead box protein P3 (Foxp3) were analyzed via the GEPIA database. Multiplex immunofluorescence and flow cytometry were performed to detect the infiltration of ILC2s and Treg cells in tissues and peripheral blood. ELISA, immunohistochemistry (IHC) and WB were carried out to verify the expression of AREG, Foxp3 and IL-10, followed by correlation analysis among ILC2s, AREG, Treg cells and IL-10. For in vitro experiments, ILC2s and peripheral blood mononuclear cells (PBMC) were isolated from cervical cancer patients and treated with recombinant human IL-33 (rhIL-33), anti-IL-33 antibody (α-IL-33), recombinant human AREG (rhAREG) and anti-human AREG antibody (α-AREG), respectively. CCK-8 assay and flow cytometry were used to evaluate the effects of different concentrations of rhAREG on the proliferation and apoptosis of HeLa and SiHa cells. The concentrations of AREG and IL-10 in cell culture supernatants were measured by ELISA, and the proportion of Treg cells were determined by flow cytometry. In addition, this study also compared the differences in the levels of ILC2s, AREG, Treg cells, and IL-10 in the peripheral blood of cervical cancer patients before and after surgery. Results: The infiltration of ILC2 and expression of AREG in tissues and peripheral blood of cervical cancer patients were markedly higher than those in the normal control group. The proportion of Treg cells and expression levels of Foxp3 and IL-10 were also significantly upregulated in cervical cancer patients (P < 0.05). Correlation analysis confirmed a positive correlation among ILC2s, AREG, Treg cells and IL-10. In vitro experiments showed that different concentrations of rhAREG had no significant effects on the proliferation or apoptosis of HeLa and SiHa cells (P > 0.05). rhIL-33 effectively activated ILC2s and upregulated AREG secretion (P < 0.01), which was reversed by α-IL-33 treatment (P < 0.05). rhAREG significantly promoted Treg cell differentiation and IL-10 secretion (P < 0.001), whereas α-AREG markedly reversed these biological effects (P < 0.01). In addition, the levels of ILC2s, Treg cells, AREG and IL-10 in peripheral blood of cervical cancer patients were significantly decreased after surgery (P < 0.0001). Conclusion: The aberrant activation of the ILC2-AREG-Treg immunoregulatory axis may participate in cervical cancer progression by shaping an immunosuppressive tumor microenvironment.

国家自然科学基金（82160545，82460502）；省部共建中亚高发病成因与防治国家重点实验室基金 （SKL-HIDCA-2024-GJ2）