小鼠胰腺癌原位灶和肝转移灶类器官的建立  

Establishment of mouse organoids from pancreatic cancer in situ and liver metastasis

作　　者：张艺璇 唐嘉彤 张舒[1] 吕瑛[1] 邹晓平[1] Zhang Yixuan;Tang Jiatong;Zhang Shu;Lyu Ying;Zou Xiaoping(Department of Gastroenterology,Nanjing Drum Tower Hospital,The Affiliated Hospital of Nanjing University Medical School,Nanjing 210008,China;School of Life Sciences,Nanjing University,Nanjing 210008,China)

机构地区：[1]南京大学医学院附属鼓楼医院消化内科,南京210008 [2]南京大学生命科学学院,南京210008

出　　处：《中华胰腺病杂志》2024年第4期265-269,共5页Chinese Journal of Pancreatology

基　　金：国家自然科学基金(81871947、82072652)。

摘　　要：目的构建小鼠胰腺癌原发灶和肝转移灶成对类器官培养体系,探讨其形态学及生物学行为。方法取6~8周龄C57BL/6小鼠,将携带荧光素酶的胰腺癌细胞PANC02注射入胰腺体尾部,待小动物活体成像仪监测到肝转移灶形成后处死小鼠,取出成对的胰腺癌原发灶和肝转移灶于体外类器官培养体系中培养。倒置相差显微镜下观察类器官的形成过程,苏木精-伊红染色观察类器官的形态结构;免疫组织化学和免疫荧光染色法检测类器官上皮细胞标志物CK19和增殖标志物Ki67蛋白的表达;蛋白质免疫印迹法和免疫组织化学检测类器官侵袭性标志物N-cadherin、E-cadherin、vimentin、snail、MMP9的表达;CellTiter-Glo®3D Cell Viability Assay检测类器官对吉西他滨的药物敏感性,计算类器官的50%抑制浓度(IC_(50))。结果成功构建了小鼠胰腺癌自发肝转移模型,并建立了胰腺癌原发灶和肝转移灶成对类器官培养体系。类器官呈球样生长,体外可连续传代达10代。肝转移灶类器官和胰腺癌原发灶类器官均呈管腔样结构,均表达上皮细胞标志物CK19和增殖标志物Ki67,且肝转移灶Ki67的阳性比例显著高于胰腺癌原发灶。肝转移性类器官侵袭性标志物N-cadherin、vimentin、snail、MMP9的表达水平显著高于胰腺癌原发灶类器官,而E-cadherin表达水平则显著低于胰腺癌原发灶类器官。肝转移灶类器官对吉西他滨的IC_(50)值为165.0 nM,高于胰腺癌原发灶类器官的108.5 nM。结论成功建立了可以稳定传代的小鼠胰腺癌原发灶和肝转移性类器官。Objective To establish a paired organoid culture system for primary pancreatic cancer lesions and liver metastatic lesions in mice,and to investigate their morphological and biological behaviors.Methods C57BL/6 mice aged 6 to 8 weeks were selected.Pancreatic cancer PANC02 cells carrying luciferase were injected into the pancreatic tail.After monitoring the formation of liver metastases using an in vivo imaging system,mice were sacrificed,and paired primary pancreatic cancer lesions and liver metastatic lesions were extracted and cultured in an in vitro organoid culture system.The formation process of organoids was observed under an inverted phase-contrast microscope.Hematoxylin and eosin staining was used to examine the morphological structure of the organoids.The expression of epithelial cell marker CK19 and proliferation marker Ki67 in the organoids was detected by immunohistochemistry and immunofluorescence staining.The expression of invasion markers N-cadherin,E-cadherin,vimentin,snail,and MMP9 was assessed by Western blotting and immunohistochemistry.The drug sensitivity of organoids to gemcitabine was evaluated using the CellTiter-Glo®3D Cell Viability Assay,and the half-maximal inhibitory concentration(IC_(50))of the organoids was calculated.Results A spontaneous liver metastasis model of pancreatic cancer in mice was successfully established,along with a paired organoid culture system for primary pancreatic cancer lesions and liver metastatic lesions.The organoids grew in a spherical shape and could be passaged up to 10 generations in vitro.Both liver metastatic lesion organoids and primary pancreatic cancer lesion organoids exhibited lumen-like structures,expressed the epithelial cell marker CK19,and the proliferation marker Ki67,with a significantly higher positive ratio of Ki67 in the liver metastatic lesions compared to the primary pancreatic cancer lesions.The expression levels of invasion markers N-cadherin,vimentin,snail,and MMP9 were significantly higher in liver metastatic organoids than in prim

关 键 词：体外培养技术 类器官 肿瘤转移 

分 类 号：R735.9[医药卫生—肿瘤]