C57BL/6新生小鼠坏死性小肠结肠炎模型改建与评估

Improvement and assessment of a neonatal murine model of necrotizing enterocolitis

目的 改建C57BL/6新生小鼠坏死性小肠结肠炎（necrotizing enterocolitis,NEC）模型,为深入探讨NEC的发病机制提供实验基础.方法 2日龄C57BL/6新生小鼠随机分为NEC模型组（36只）和对照组（11只）,NEC模型组给予100％氮气90s＋4℃10 min＋脂多糖7 mg/kg干预建模,对照组仅予以同等体积生理盐水.比较两组间生长发育、肠道大体及病理差异,荧光定量PCR检测相关基因mRNA变化,免疫组化方法评估肠道细胞迁移和自我更新,免疫荧光检测细胞凋亡和增殖.Nadler标准病理评分≥3分定义为NEC样肠道损伤.结果 NEC模型组24 h之内死亡1只,48 h存活率为81％,72 h存活率为69％.对照组存活率为100％.NEC模型组和对照组实验前体重差异无统计学意义.实验24 h、48 h和72 h,模型组平均体重均显著低于对照组,P＜0.05.模型组新生小鼠活动减少甚者完全静止,对刺激反应欠佳或无反应;而对照组生长发育良好[临床评分：对照组中位数=0（0～1）,NEC模型组中位数=7（3～12）,P＜0.01].NEC模型组肠道标本表现为全肠段或部分肠段不同程度扩张和肠壁积气,严重者呈串珠样;以回盲部及其近端病变更为严重[肠道评分：对照组中位数=0（0～1）;NEC模型组中位数=4（1～6）,P＜0.01],镜下肠绒毛病变明显,Nadler标准病理评分≥3分20例,成模率为71％（20/28）.与对照组相比,NEC模型组细胞增殖减少（P＜0J.05）,细胞凋亡增加（P＜0.05）,细胞迁移距离缩短（P＜0.05）,炎症相关基因ICAM、CXCL-1、CX-CL-2表达上调,肠道干细胞Lgr5表达下降.结论 本研究成功改建C57BL/6新生小鼠NEC模型,模型简洁高效、稳定可重复,能用于NEC的进一步研究.

Objective To improve and evaluate the model of necrotizing enterocolitis （NEC） in C57BL/6 neonatal mice so as to provide experimental rationales for further pathogenic studies of NEC.Methods Two-day-old C57BL/6 neonatal mice were randomly divided by a ratio of 3..1 into NEC （n =36） and control （n =11） groups.The NEC group was stressed by hypoxia and hypothermia plus an intraperitoneal injection of lipopolysaccharide （LPS） while the control group had no stress factors.The inter-group differences of clinical status,macroscopic gut and histology were compared.Histological scores of 3 or 4 were considered to have developed NEC.Results NEC was present in 71％ of NEC group.Control mice showed no evidence of NEC.Clinical sickness score was higher in NEC group （median =7;range =3-12） than controls （median =0；range =0-1;P＜0.01）.There were decreases in proliferative activity and cell migration and enterocyte apoptosis intensified.The expressions of ICAM,CXCL-1,CXCL-2 and Lgr5 were significantly different from those in controls.Conclusions The study has established a stable and reliable 2-day-old C57BL/6 murine model of NEC through hypoxia,hypothermia and LPS.And it may be used for further studies of NEC.