目的 采用气液交互法构建患者来源的结直肠癌类器官，探索该模型在扶正类中药成分免疫活性评价中的适用性。方法 选自南京中医药大学附属南京中医院肛肠中心收治入院的结直肠癌患者术中肿瘤组织，运用气液交互法构建结直肠癌类器官模型；采用苏木素-伊红（hematoxylin-eosin，HE）染色检测结直肠癌组织标本与类器官样本肿瘤组织的形态特点；采用免疫组化法检测细胞角蛋白20（cytokeratin 20，CK20）、重组Ki-67蛋白（recombinant Ki-67 protein，Ki-67）、增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）表达水平；采用流式细胞术检测白术多糖、茯苓多糖、香菇多糖及不同浓度的白术-茯苓多糖联用干预对类器官肿瘤细胞活性及免疫细胞比例的影响。结果 患者来源的结直肠癌类器官中含有T细胞与B细胞，白术多糖、茯苓多糖、香菇多糖能够提升类器官肿瘤微环境中B细胞的比例，且白术-茯苓多糖联合使用时，效果更显著。结论 采用气液交互法构建患者来源的结直肠癌类器官能够应用于扶正类中药活性多糖的药效评价，该模型可为扶正类中药对结直肠癌免疫微环境的调节作用研究提供理论依据及方法学参考。

Objective To construct organoids derived from colorectal cancer patients using the air-liquid interaction method, the applicability of this model in evaluating immune activity of components of “Fuzheng” traditional Chinese medicine was explored. Methods Intraoperative colorectal cancer tumor tissues were obtained from patients admitted to the Anorectal Center of Nanjing Hospital of Chinese Medicine. Organoids from colorectal cancer were constructed by air-liquid interaction method. Hematoxylin-eosin (HE) staining was used to detect morphological characteristics of colorectal cancer tissue samples and organoids. The expression levels of cytokeratin 20 (CK20), recombinant Ki-67 protein (Ki-67), proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were detected by immunohistochemistry. Flow cytometry was used to detect the effects of Atractylodes macrocephala, Poria cocos, Lentinus edodes polysaccharides and different concentrations of A. macrocephala koidz and P. cocos polysaccharides combined on the activity of organoid tumor cells and the proportion of immune cells. Results The patient-derived colorectal cancer organoids contained both T cells and B cells. Treatment with A. macrocephala, P. cocos, L. edodes polysaccharides led to an increase in the proportion of B cells within the tumor microenvironment of the organoids. This effect was more pronounced when the two polysaccharides, A. macrocephala and P. cocos polysaccharides, were combined. Conclusion The air-liquid interaction method can be applied to construct patient-derived colorectal cancer organoids for evaluating the efficacy of active polysaccharides from “Fuzheng” traditional Chinese medicine. This model can provide theoretical and methodological support for further study of the regulatory effects of “Fuzheng” traditional Chinese medicine on the immune microenvironment of colorectal cancer.

R285.5

江苏省重点研发计划(社会发展)项目(BE2021611);南京中医药大学自然科学基金项目(XZR2021044)