摘要
目的比较不同肠上皮细胞株在3D培养条件下形成肠类器官的潜能及特点,为利用肠上皮细胞株培养肠类器官提供实验研究基础。方法使用IEC-6、CT26.WT、Caco-2三种肠上皮细胞株进行3D培养,分析不同细胞株形成肠类器官的潜能。流式分选获取Caco-2单细胞后使用Matrigel进行培养并测定其生长速度。比较2D和3D培养条件下Caco-2细胞中肠道干细胞标志物Lgr5、Olfm4及增殖标志物Ki67的mRNA表达差异。免疫荧光染色分析2D和3D培养时Caco-2细胞内Ki67阳性细胞及lamp1阳性溶酶体的分布特点。比较5 Gy和15 Gy照射后Caco-2来源肠类器官中TUNEL阳性细胞分布特点。结果 3D培养时,Caco-2细胞可形成肠类器官,而IEC-6和CT26.WT细胞不能形成肠类器官,且单个Caco-2细胞也可形成肠类器官。Caco-2来源肠类器官中肠道干细胞标志物Lgr5、Olfm4和细胞增殖标志物Ki67的mRNA表达水平较2D培养时显著降低(P<0.05),Ki67阳性增殖细胞较2D培养显著减少。Caco-2来源肠类器官中细胞内溶酶体呈极性分布,与小肠组织切片中溶酶体在肠上皮细胞内的分布一致。电离辐射可引起Caco-2来源肠类器官内细胞发生凋亡,且凋亡细胞比例与照射剂量密切相关。结论 Caco-2在3D培养时可形成与在体肠道结构和功能类似的肠类器官,为下一步进行胃肠道电离辐射研究和药物评价研究提供了更加稳定可靠的体外研究模型。
Objective To compare the potentiality and characteristics of different intestinal epithelial cell(IEC) lines to grow into enteroids when cultured in 3 D system in order to provide experimental basis for the cultivation of enteroids by IECs. Methods Three IEC lines, IEC-6, CT26.WT and Caco-2, were cultured in a 3D system, and further characterized by morphology. Single Caco-2 cell was acquired by fluorescent activated cell sorting(FACS) and seeded in Matrigel. The growth speed of enteroids derived from single Caco-2 cell was determined. The expression levels of intestinal stem cell(ISC) genes, including Lgr5 and Olfm4, and proliferative marker, Ki67 were determined by qRT-PCR, and the results were compared between 2D and 3D cultured Caco-2 cells. The distribution of Ki67-positive cells and lamp1-positive lysosomes in 2D and 3D cultured Caco-2 cells were observed with aid of immunofluorescent staining. TUNEL staining was employed to analyze the apoptosis of Caco-2 cells after 5 and 15 Gy radiation, respectively. Results Among the 3 IEC lines, only Caco-2 cells could grow into enteroids when cultured in 3D system. Single Caco-2 cell acquired by FACS could also form enteroids. Compared to 2D culture, Caco-2 derived enteroids expressed lower mRNA levels for ISC markers Lgr5 and Olfm4, and also lower proliferative marker Ki67(P<0.05). There were less Ki67-positive cells in 3D than 2D cultured Caco-2 cells. Lysosomes in 3D cultured Caco-2 cells exhibited a polar distribution, which was similar to them in in vivo pattern. Radiation exposure caused apoptosis of IECs in Caco-2 derived enteroids, and the apoptotic rate was positively correlated to the dose of radiation. Conclusion Caco-2 cells could form enteroids when cultured in 3D system, which is similar to the structure of in vivo intestine in structure and function. This study provides reliable in vitro model for gastrointestinal radiation and pharmaceutical evaluation in the future.
作者
欧静
徐珍妮
刘登群
王涛
王锋超
王军平
粟永萍
OU Jing;XU Zhenni;LIU Dengqun;WANG Tao;WANG Fengchao;WANG Junping;SU Yongping(State Key Laboratory of Trauma,Burns and Combined Injury,Department of Anti-radiation Medicine,Chongqing Engineering Research Center for Nanomedicine,Institute of Combined Injury,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,40038,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2020年第1期31-38,共8页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81371688,81673089,81874255)
重庆市基础科学与前沿技术研究计划项目(CSTC2013jcyjA10038)
创伤、烧伤与复合伤国家重点实验室自主研究课题(SKLZZ201634).
作者简介
通信作者:刘登群,E-mail:dengqunliu@163.com
