Establishment of small intestinal organoid models in a novel culture system
10.3760/cma.j.cn311367-20230503-00206
- VernacularTitle:新型培养体系下小肠类器官模型的构建
- Author:
Bian WU
1
;
Guili LIANG
;
Chengfeng XING
;
Zaozao WU
;
Junmo WU
;
Yu KANG
;
Yinglei MIAO
;
Danfeng LAN
Author Information
1. 云南省第一人民医院普外二科，昆明 650032
- Keywords:
New small intestinal organoids;
Intestinal epithelial regeneration;
8C culture system;
ENR culture system;
Tumor necrosis factor-alpha
- From:
Chinese Journal of Digestion
2023;43(11):764-770
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a new type of small intestinal organoids with injury-related regenerative capacity, and to simulate the process of intestinal injury, regeneration, and repair in vitro. Methods:The crypt structures of ileal mucosa from 6 to 8 weeks old, 18 to 24 g specific pathogen-free C57BL/6 mice were isolated. The ENR, ENR+ tumor necrosis factor-α(TNF-α) and 8C culture systems were designed to establish small intestinal organoids under conditions of intestinal homeostasis, inflammatory injury and injury-related regeneration, and the morphology of intestinal organoids were observed. The cell types and spatial arrangements of intestinal organoids, and the expression of genes clusterin( Clu), annexin A1( Anxa1), stem cell antigen-1( Sca1) and regenerating islet-derived protein 3-beta( Reg3 b) at protein levels were detected by immunofluorescence staining. The expression of genes Clu, Anxa1, Sca1 and Reg3 b at mRNA levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Independent sample- t test was used for statistical analysis. Results:In ENR and 8C culture system, both intestinal organoids contained intestinal stem cells, goblet cells, Paneth cells and intestinal endocrine cells, and the spatial arrangement of cells was similar to the intestinal epithelium. In the 8C culture system, the amplification capacity of the new small intestinal organoids was significantly enhanced, the growth rate was faster, and the structure was larger and more complex than those of small intestinal organoids in ENR and ENR+ TNF-α culture systems. The results of qRT-PCR showed that, the relative mRNA expression levels of novel small intestinal organoid regeneration genes Clu, Anxa1, and Sca1 in the 8C culture system were higher than those in the ENR and ENR+ TNF-α culture systems (0.68±0.31 vs.0.20±0.07 and 0.36±0.19, 0.48±0.13 vs. 0.07±0.02 and 0.18±0.11, 0.56±0.20 vs. 0.02±0.01 and 0.08±0.04), and the differences were statistically significant ( t=4.82 and 2.77, 8.62 and 4.89, and 8.58 and 7.50; all P<0.05). The results of immunofluorescence staining indicated that, the expression levels of novel small intestinal organoid regeneration genes Clu, Anxa1, Sca1 and Reg3 b at protein level in the 8C culture system were higher than those in the ENR and ENR+ TNF-α culture systems (31.62±1.69 vs. 9.73±2.39 and 15.11±2.16, 42.65±1.85 vs. 19.70±1.18 and 24.97±2.82, 63.80±2.73 vs. 37.10±1.59 and 43.27±2.53, 53.26±1.84 vs. 27.75±3.78 and 33.16±3.50), and the differences were statistically significant( t=12.95 and 10.41, 18.13 and 9.09, 14.63 and 9.56, and 10.51 and 8.80; all P<0.001). Conclusion:The small intestinal organoids established in the novel culture system have the characteristics of injury-related regeneration, and provide a novel in vitro model for studying the regeneration of epithelial tissues and organs.
