Xu Lingqi,Ma Shurong,Chen Lulu,et al.Improvements and evaluations of animal models of neonatal necrotizing enterocolitis[J].Journal of Clinical Pediatric Surgery,2023,22(06):569-575.[doi:10.3760/cma.j.cn101785-202202036-014]
坏死性小肠结肠炎动物模型建立方法的改进与评价
- Title:
- Improvements and evaluations of animal models of neonatal necrotizing enterocolitis
- Keywords:
- Enterocolitis; Necrotizing; Models; Animal; Feasibility Studies
- 摘要:
- 目的 目前新生儿坏死性小肠结肠炎（necrotizing enterocolitis，NEC）动物模型的构建方式仍不统一，本研究旨在明确更贴切临床NEC患儿实际情况的动物建模方式。方法 随机将54只C57BL/6新生小鼠分为五组，依次为：对照组（Ctrl组，10只）、缺氧+人工喂养组（HF组，10只）、缺氧+人工喂养+冷刺激组（Cold组，12只）、缺氧+人工喂养+脂多糖（lipopdysaccharide，LPS）组（LPS组，11只）、缺氧+人工喂养+NEC肠内细菌组（Bac组，11只）；分别建立NEC动物模型后，评估肠道组织病理、NEC相关肠上皮屏障蛋白（β-catenin、Occludin）和肠上皮细胞死亡标记性分子（CC3、RIPK1、PARP1）以及促炎细胞因子（IL-6、TNF-α、MCP1）的表达变化。结果 按照肠道组织学Nadler评分≥ 2分视为NEC样肠道损伤。本研究除Ctrl组和HF组外，其余三组NEC造模的肠道组织病理均满足NEC样肠道损伤标准。与HF组（30%）、Cold组（83.3%）和LPS组（81.8%）相比，Bac组的建模成功率最高（100%），且造模期间Bac组小鼠的精神状况、腹胀腹泻、活动度等与NEC临床情况更为贴切，同时Bac组小鼠的肠道屏障蛋白β-catenin和Occludin表达下降，与Ctrl组相比，差异有统计学意义（P<0.05）。LPS组和Bac组肠上皮细胞死亡标记性分子RIPK1和PARP1表达上调，炎症因子IL-6、TNF-α和MCP1表达水平增加，与Ctrl组相比，差异均有统计学意义（P<0.05）。结论 本研究成功比较了4种NEC动物模型的建立方法，明确了一种更贴近临床NEC患儿实际情况的动物建模方法，即"缺氧+人工喂养+NEC肠内细菌"。该造模方法成功率高，肠道组织病理损伤、肠道屏障蛋白表达和全身炎症反应与临床上NEC患儿特征相似度更高。
- Abstract:
- Objective The construction of animal models of neonatal necrotizing enterocolitis (NEC) is still not uniform, and animal modeling approaches that are more relevant to the actual clinical situation of NEC childern should be clarified.Methods Fifly-four newborn mice were randomized into five groups of control (Ctrl), hypoxia plus artificial feeding (HF), hypoxia plus artificial feeding plus cold stimulation (Cold), hypoxia plus artificial feeding plus lipopolysaccharide (LPS) and hypoxia plus artificial feeding plus intestinal bacteria in NEC (Bac).After successful modeling, intestinal pathology, NEC-related intestinal epithelial barrier proteins (β-catenin & Occludin), intestinal epithelial cell death (CC3, RIPK1 & PARP1) and pro-inflammatory cytokines (IL-6, TNF-α & MCP1) were evaluated.Results Nadler score ≥ 2 according to intestinal histology was considered as NEC-like intestinal injury. In this study, the intestinal histopathology of the three NEC-modeled groups met the criteria for NEC-like intestinal injury, except for the Ctrl and HF groups. Compared with the NEC modeling groups HF (30%), Cold (83.3%) and LPS (81.8%), the Bac group had the highest modeling success rate (100%), and the mental status, bloating and diarrhea, and mobility of the mice in the Bac group during the modeling period were more consistent with clinical NEC. Meanwhile, the expression of intestinal barrier proteins β-catenin and Occludin was decreased in the Bac group mice, and the difference was statistically significant compared with the Ctrl group (P<0.05). the expression of intestinal epithelial cell death marker molecules RIPK1 and PARP1 was upregulated in the LPS and Bac groups, and the expression levels of inflammatory factors IL-6, TNF-α and MCP1 were increased compared with the Ctrl group, with statistically significant differences (P<0.05). Conclusion This study has successfully established four NEC animal models and verified a more appropriate animal modeling method of clinical NEC, namely "hypoxia plus artificial feeding plus NEC intestinal bacteria".Such a modeling method has a high success rate.And intestinal histopathological injury, intestinal barrier protein expression and systemic inflammatory response mimic closely the clinical characteristics of NEC.
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备注/Memo
收稿日期:2022-02-07。
基金项目:江苏省卫生健康委医学科研项目（H2019002）
通讯作者:汪健,Email:wj196312@vip.163.com