小鼠肠上皮细胞岩藻糖基化与新生儿坏死性小肠结肠炎发生的关系

Relations of the fucosylation of intestinal epithelial cells to the onset of neonatal necrotizing enterocolitis

目的观察肠上皮细胞岩藻糖基化水平在新生儿坏死性小肠结肠炎(NEC)小鼠模型中的变化,探讨其可能的机制。方法将42只10日龄的C57BL/6新生小鼠按随机数字表法分为NEC组(n=21,采用人工喂养+缺氧+冷刺激方法建立NEC模型)和对照组(n=21,母鼠喂养,不做处理),建模3d后处死。采用NEC病理损伤评分评估建模效果;采用流式细胞技术检测岩藻糖基化肠上皮细胞(F-ECs)和肠道固有层3型天然淋巴细胞(ILC3s)比例;采用实时荧光定量PCR检测肠上皮细胞白细胞介素-22受体(IL-22R)、岩藻糖基转移酶2(Fut2)和固有层淋巴细胞白细胞介素-22(IL-22)的表达水平;采用ELISA法检测固有层淋巴细胞IL-22蛋白的表达水平。结果成功建立NEC小鼠模型。流式细胞检测结果显示,NEC模型组F-ECs百分比低于对照组,差异有统计学意义(P<0.05);NEC模型组肠道固有层ILC3s百分比也明显低于对照组,差异有统计学意义(P<0.001)。实时荧光定量PCR结果显示,NEC模型组上皮细胞IL-22R、Fut2 mRNA表达水平明显低于对照组(P<0.001),固有层淋巴细胞IL-22 mRNA表达水平也明显低于对照组,差异有统计学意义(P<0.01)。NEC模型组固有层淋巴细胞IL-22蛋白表达水平低于对照组,差异有统计学意义(P<0.001)。结论肠上皮细胞岩藻糖基化参与了NEC小鼠模型的发病,其机制可能与ILC3s-IL-22-Fut2轴有关。

Objective To investigate the changes of fucosylation level of intestinal epithelial cells in the mouse model of neonatal necrotizing enterocolitis(NEC), and explore it’s possible mechanism. Methods Forty-two 10-day old C57 BL/6 mice were randomly divided into NEC group(n=21, NEC model was established by artificial feeding, hypoxia and cold stimulation) and control group(n=21, mother feeding and no treatment), and mice were sacrificed 3 days after modeling. NEC pathological lesion scoring was performed to assess the modeling effect. Flow cytometry was used to detect the proportion of fucosylated intestinal epithelial cells(F-ECs) and intestinal lamina propria group 3 innate lymphocytes(ILC3 s). The levels of interleukin-22 receptor(IL-22 R), fucosyltransferase 2(Fut2) of intestinal epithelial cells and interleukin-22(IL-22) of lamina propria lymphocyte were detected by quantitative real-time PCR. The level of IL-22 protein in lamina propria was detected by enzyme-linked immunosorbent assay(ELISA). Results The NEC mouse model was successfully established. Flow cytometry results showed that the percentage of F-ECs was lower in NEC model group than in control group(P<0.05). The percentage of intestinal ILC3 s was also significantly lower in NEC model group than in control group with statistically significant difference(P<0.001). The results of quantitative real-time PCR showed that the mRNA levels of IL-22 R and Fut2 were significantly lower in epithelial cells of NEC model group than in control group(P<0.001). The mRNA level of IL-22 was also significantly lower in lamina propria lymphocytes of NEC model group than in control group(P<0.01). The expression level of IL-22 protein in lamina propria lymphocytes was lower in NEC model group than in control group(P<0.001). Conclusion Decreased fucosylation of intestinal epithelial cells is involved in the pathogenesis of NEC mouse model, and the mechanism may be related to the ILC3 s-IL-22-Fut2 axis.