[1]欧静,徐珍妮,刘登群,等.3D培养体系中不同肠上皮细胞株形成肠类器官潜能的比较及应用[J].第三军医大学学报,2020,42(01):31-38.
 OU Jing,XU Zhenni,LIU Dengqun,et al.Potential capacities of different intestinal epithelial cell lines to form enteroids in 3D culture system: comparison and application[J].J Third Mil Med Univ,2020,42(01):31-38.
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第01期
页码:
31-38
栏目:
基础医学
出版日期:
2020-01-15

文章信息/Info

Title:
Potential capacities of different intestinal epithelial cell lines to form enteroids in 3D culture system: comparison and application
作者:
欧静徐珍妮刘登群王涛王锋超王军平粟永萍
陆军军医大学(第三军医大学)军事预防医学系防原医学教研室,全军复合伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆市纳米医学工程研究中心
Author(s):
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

关键词:
肠上皮细胞株Caco-2细胞肠类器官干细胞电离辐射
Keywords:
intestinal epithelial cells Caco-2 cells enteroid stem cells ionizing radiation
分类号:
R322.45; R329-33; R329.3
文献标志码:
A
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。

Abstract:

Objective To compare the potentiality and characteristics of different intestinal epithelial cell (IEC) lines to grow into enteroids when cultured in 3D system in order to provide experimental basis for the cultivation of enteroids by IECs. MethodsThree 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. ResultsAmong 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. ConclusionCaco-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.

参考文献/References:

[1]SATO T, VRIES R G, SNIPPERT H J, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche[J]. Nature, 2009, 459(7244): 262-265. DOI:10.1038/nature07935.
[2]SCHEPERS A G, SNIPPERT H J, STANGE D E, et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas[J]. Science, 2012, 337(6095): 730-735. DOI:10.1126/science.1224676.
[3]SCHMITT M, SCHEWE M, SACCHETTI A, et al. Paneth cells respond to inflammation and contribute to tissue regeneration by acquiring stem-like features through SCF/c-kit signaling[J]. Cell Rep, 2018, 24(9): 2312-2328.e7. DOI:10.1016/j.celrep.2018.07.085.
[4]STELZNER M, HELMRATH M, DUNN J C, et al. A nomenclature for intestinal in vitro cultures[J]. Am J Physiol Gastrointest Liver Physiol, 2012, 302(12): G1359-G1363. DOI:10.1152/ajpgi.00493.2011.
[5]YUAN Y H, DING D K, ZHANG N, et al. TNF-α induces autophagy through ERK1/2 pathway to regulate apoptosis in neonatal necrotizing enterocolitis model cells IEC-6[J]. Cell Cycle, 2018, 17(11): 1390-1402. DOI:10.1080/15384101.2018.1482150.
[6]XU J X, XIONG W, ZENG Z, et al. Effect of ART1 on the proliferation and migration of mouse colon carcinoma CT26 cells in vivo[J]. Mol Med Rep, 2017, 15(3): 1222-1228. DOI:10.3892/mmr.2017.6152.
[7]HIDALGO I J, RAUB T J, BORCHARDT R T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability[J]. Gastroenterology, 1989, 96(3): 736-749.
[8]WANG Z, WANG S N, XU T Y, et al. Organoid technology for brain and therapeutics research[J]. CNS Neurosci Ther, 2017, 23(10): 771-778. DOI:10.1111/cns.12754.
[9]KRUITWAGEN H S, OOSTERHOFF L A, VERNOOIJ I G W H, et al. Long-term adult Feline liver organoid cultures for disease modeling of hepatic steatosis[J]. Stem Cell Rep, 2017, 8(4): 822-830. DOI:10.1016/j.stemcr.2017.02.015.
[10]MORIZANE R, BONVENTRE J V. Kidney organoids: A translational journey[J]. Trends Mol Med, 2017, 23(3): 246-263. DOI:10.1016/j.molmed.2017.01.001.
[11]MATANO M, DATE S, SHIMOKAWA M ,et al.Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids[J]. Nat Med,2015,21(3):256-262. DOI: 10.1038/nm.3802.
[12]MADISON B B, LIU Q, ZHONG X, et al. LIN28B promotes growth and tumorigenesis of the intestinal epithelium via Let-7[J]. Genes Dev, 2013, 27(20): 2233-2245. DOI:10.1101/gad.224659.113.
[13]SATO T, CLEVERS H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications[J]. Science, 2013, 340(6137): 1190-1194. DOI:10.1126/science.1234852.

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更新日期/Last Update: 2020-01-07