新型培养体系下小肠类器官模型的构建

Establishment of small intestinal organoid models in a novel culture system

目的构建具有损伤再生能力的新型小肠类器官,体外模拟肠道损伤、再生和修复过程。方法分离6~8周龄、18~24 g无特定病原体动物级C57BL/6小鼠回肠黏膜隐窝结构,设计ENR、ENR+肿瘤坏死因子-α(TNF-α)和8C培养体系,分别构建肠道稳态、炎症损伤和损伤再生条件下的小肠类器官并观察形态。通过免疫荧光染色检测类器官包含的细胞类型和空间排布,以及损伤再生基因凝集素蛋白(Clu)、膜联蛋白A1(Anxa1)、干细胞抗原1(Sca1)和小鼠再生胰岛衍生蛋白3β(Reg3 b)的蛋白质表达情况。通过实时荧光定量聚合酶链反应(qRT-PCR)检测Clu、Anxa1、Sca1、Reg3 b的mRNA表达情况。统计学方法采用独立样本t检验。结果8C和ENR培养体系下的小肠类器官均包含肠道干细胞、杯状细胞、潘氏细胞和肠道内分泌细胞,细胞空间排布与肠上皮基本一致。8C培养体系下的新型小肠类器官的扩增能力较ENR、ENR+TNF-α培养体系下的小肠类器官明显增强,生长速度更快,结构更大、更复杂。qRT-PCR结果显示,8C培养体系下的新型小肠类器官再生基因Clu、Anxa1和Sca1的mRNA相对表达水平均高于ENR和ENR+TNF-α培养体系(0.68±0.31比0.20±0.07、0.36±0.19,0.48±0.13比0.07±0.02、0.18±0.11,0.56±0.20比0.02±0.01、0.08±0.04),差异均有统计学意义(t=4.82、2.77,8.62、4.89,8.58、7.50;均P<0.05)。免疫荧光检测显示,8C培养体系下的新型小肠类器官再生基因Clu、Anxa1、Sca1和Reg3 b的蛋白质表达水平均高于ENR、ENR+TNF-α培养体系(31.62±1.69比9.73±2.39、15.11±2.16,42.65±1.85比19.70±1.18、24.97±2.82,63.80±2.73比37.10±1.59、43.27±2.53,53.26±1.84比27.75±3.78、33.16±3.50),差异均有统计学意义(t=12.95、10.41,18.13、9.09,14.63、9.56,10.51、8.80;均P<0.001)。结论新型培养体系下建立的小肠类器官具备损伤再生特征,为研究上皮组织器官再生提供了新的体外模型。

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-αculture 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-αculture 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-αculture 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.