肿瘤相关成纤维细胞促胃癌类器官增殖与耐药的初步研究  

Cancer-associated fibroblasts promote proliferation and drug resistance of gastric cancer organoids:a primary study

作　　者：张媛媛 段振铨 李雨贤 黄梦秋 朱宝行 邱远 邹全明[2] 彭六生[2] 马代远[1] ZHANG Yuanyuan;DUAN Zhenquan;LI Yuxian;HUANG Mengqiu;ZHU Baohang;Qiu Yuan;ZOU Quanming;PENG Liusheng;MA Daiyuan(Department of Oncology,Affiliated Hospital of North Sichuan Medical College,Nanchong,Sichuan;Department of Microbiology and Biochemical Pharmacy,Faculty of Pharmacy and Laboratory Medicine,Army Medical University(Third Military Medical University),Chongqing;Department of General Surgery,Second Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,China)

机构地区：[1]川北医学院附属医院肿瘤科,四川南充 [2]陆军军医大学(第三军医大学)药学与检验医学系微生物与生化药学教研室,重庆 [3]陆军军医大学(第三军医大学)第二附属医院普外科,重庆

出　　处：《陆军军医大学学报》2025年第5期453-461,共9页Journal of Army Medical University

基　　金：北京科创医学发展基金会(KC2023-JX-0186-FM057);川北医学院附属医院科研发展计划(2023PTZK014)。

摘　　要：目的体外构建胃癌类器官与肿瘤相关成纤维细胞的共培养模型,利用该模型探究肿瘤相关成纤维细胞对胃癌类器官增殖与化疗耐药的影响。方法收集于2023年2月至2024年3月在陆军军医大学第二附属医院普外科接受手术切除的12例胃癌患者的肿瘤组织。采用体外三维构建胃癌类器官;通过HE染色进行形态学分析,采用免疫组织化学染色检测细胞角蛋白CK7、癌胚抗原CEA和增殖相关因子Ki-67的表达。培养同一患者来源的肿瘤相关成纤维细胞,通过光镜观察分析其形态;采用流式细胞染色检测其表型。将胃癌类器官与肿瘤相关成纤维细胞按1∶1比例共培养,相差显微镜下观察胃癌类器官的生长情况并分析其数量、平均直径与总面积。将单独培养的类器官设置为对照组,在对照组和共培养组中加入化疗药物5-氟尿嘧啶和奥沙利铂处理48 h,利用CellTiter-Glo^(R)3D试剂盒和CellEvent^(TM)Caspase 3/7分别检测类器官的细胞活力与凋亡情况。结果成功构建出10例患者来源的胃癌类器官与肿瘤相关成纤维细胞,胃癌类器官表现出与原发肿瘤一致的形态学特征并在CK7、CEA与Ki-67的表达上具有相似性。肿瘤相关成纤维细胞呈现出典型的梭状及CD326^(-)、CD45^(-)、CD31^(-)、α-SMA^(+)、CD73^(+)、CD90^(+)、CD105^(+)表型特征。与单独培养的类器官对照组比较,与肿瘤相关成纤维细胞共培养组的类器官形成数量更多、平均直径更长且总面积更大(P<0.05)。在化疗药物5-氟尿嘧啶和奥沙利铂处理下,对照组的半数抑制浓度分别为10.66和3.26μmol/L,而共培养组则分别为46.23和91.11μmol/L;与类器官单独培养的对照组比较,共培养组中类器官的CellEvent?Caspase 3/7阳性凋亡细胞明显减少。结论与单独培养的类器官比较,肿瘤相关成纤维细胞共培养模型能更好地模拟体内肿瘤微环境的促瘤增殖与耐药效应。Objective To construct an in vitro co-culture model of gastric cancer organoids and cancer-associated fibroblasts(CAFs),and investigate the role of cancer-associated fibroblasts in the proliferation and chemotherapy resistance of gastric cancer organoids.Methods Tumor tissues from 12 gastric cancer patients undergoing surgical treatment in Department of General Surgery of Second Affiliated Hospital of Army Medical University from February 2023 to March 2024 were collected to construct gastric cancer organoids using 3D culture.HE staining was used to observe the morphology,and immunohistochemical assay was employed to determine the expression of cytokeratin CK7,carcinoembryonic antigen(CEA),and proliferation marker Ki-67.After CAFs derived from the same patient were cultured,observed for their morphology under a light microscope,and detected for the phenotype by flow cytometry,the cells were co-cultured with gastric cancer organoids in a 1:1 ratio.Phase-contrast microscopy was applied to observe the growth of the organoids and analyze the number,average diameter,and total area.Then,organoids cultured alone served as the control group.After the control and co-culture groups were treated with chemotherapy drugs,5-fluorouracil and oxaliplatin,for 48 h,the viability and apoptosis of organoids were assessed with CellTiter-Glo^(R)3D assay and CellEvent^(TM)Caspase 3/7 activity,respectively.Results Gastric cancer organoids and CAFs were successfully established from 10 gastric cancer patient-derived samples.The gastric cancer organoids exhibited morphological characteristics consistent with the corresponding primary tumors,and showed positive expression of CK7,CEA,and Ki-67.CAFs displayed typical spindle-shaped morphology and exhibited the phenotypic markers CD326^(-),CD45^(-),CD31^(-),α-SMA^(+),CD73^(+),CD90^(+),and CD105^(+).Compared to the organoids cultured alone,the organoids co-cultured with CAFs showed more formation of organoids,in larger average diameter,and taking larger total area(P<0.05).After the treatment

关 键 词：胃癌 类器官 成纤维细胞 共培养模型 药物敏感检测 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R73-354[医药卫生—基础医学] R735.2