| 摘要: |
| 目的 通过构建核因子ⅠB（NFIB）条件性基因敲除（cKO）小鼠，探讨髓系细胞NFIB的表达与肠道炎症的关系。方法 利用人类蛋白质图谱数据库、基因型-组织表达数据库和FANTOM5数据库查找NFIB在炎症细胞中的表达情况。运用CRISPR/Cas9技术构建NFIB-flox小鼠，并与Lyz2-Cre转基因小鼠杂交，将后代自交获得髓系特异性NFIB cKO小鼠（NFIBfl/flLyz2-Cre小鼠）。经琼脂糖凝胶电泳鉴定小鼠基因型后，选取C57BL/6N品系的NFIB cKO小鼠4只为实验组，非cKO小鼠4只为对照组。两组小鼠均使用2.5%葡聚糖硫酸钠盐以相同条件诱导，构建慢性结肠炎模型，从临床表现和组织病理学方面评估结肠炎严重程度。结果 经分析发现NFIB在髓系细胞来源的粒细胞、单核细胞中均有表达，且在中性粒细胞中高表达。成功地利用CRISPR/Cas9技术和Cre-loxP系统构建了髓系特异性NFIB cKO小鼠。葡聚糖硫酸钠盐诱导的肠炎模型NFIB cKO小鼠在短时间内出现腹泻、肉眼血便、活动减少、体重减轻等情况。肠道大体观察显示NFIB cKO小鼠结肠较非cKO小鼠缩短［（8.23±0.35） cm vs （10.30±0.36） cm，P＜0.01］。肠H-E染色显示NFIB cKO小鼠肠黏膜腺结构改变和结缔组织增生伴广泛炎症细胞浸润，NFIB cKO小鼠的组织学评分高于非cKO小鼠［（4.25±0.50）分vs（0.50±0.58）分，P＜0.01］。肠免疫组织化学染色结果显示，CD11b阳性细胞在NFIB cKO小鼠较非cKO小鼠中募集更多。结论 本实验成功构建了髓系特异性NFIB cKO小鼠，并发现髓系细胞中的NFIB能够减轻免疫细胞（粒细胞或/和单核细胞）浸润，抑制肠道炎症。 |
| 关键词: 条件性基因敲除 核因子IB 肠道炎症 髓系细胞 动物模型 |
| DOI：10.16781/j.CN31-2187/R.20210488 |
| 投稿时间：2021-05-10修订日期：2022-02-21 |
| 基金项目:国家自然科学基金（81972285），肿瘤免疫病理学教育部重点实验室开发课题（2022jsz808）. |
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| Construction of myeloid specific nuclear factor ⅠB conditional gene knockout mice and its intestinal inflammation manifestation |
| HU Manqiu,ZHOU Li,CHEN Siyuan,LIU Hongtao,ZHANG Hao,HE Song,ZHOU Zhihang* |
(Department of Gastroenterology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China
*Corresponding author) |
| Abstract: |
| Objective To investigate the relationship between the expression of nuclear factor ⅠB (NFIB) in myeloid cells and intestinal inflammation by constructing NFIB conditional gene knockout (cKO) mice. Methods Human Protein Atlas database, Genotype-Tissue Expression database, and FANTOM5 database were used to investigate the expression of NFIB in inflammatory cells. NFIB-floxed mice were constructed using CRISPR/Cas9 technology and hybridized with LyZ2-Cre transgenic mice. Myeloid specific NFIB cKO mice (NFIBfl/flLyz2-Cre) were obtained by self-crossing the progeny. After the genotype identification of mice by agarose gel electrophoresis, 4 NFIB cKO mice of C57BL/6N strain were selected as experimental group, and 4 non-cKO mice were selected as control group. Both groups were induced with 2.5% dextran sulfate sodium salt (DSS) under the same condition to establish a chronic colitis model, and the severity of colitis was evaluated by clinical manifestations and histopathology. Results Analysis showed that NFIB was expressed in both myeloid granulocytes and monocytes, and the highest expression was found in neutrophils. NFIB cKO mice were successfully constructed using CRISPR/Cas9 technology and Cre-loxP system. DSS-induced enteritis NFIB cKO mice developed diarrhea, gross blood stools, reduced activity, and weight loss in a short time. The gross examination of the intestines showed that the colon of the NFIB cKO mice was significantly shorter than that of the non-cKO mice (［8.23±0.35］ cm vs ［10.30±0.36］ cm, P＜0.01). Intestinal H-E staining showed changes in mucosal glandular structure and connective tissue hyperplasia with extensive inflammatory cell infiltration in NFIB cKO mice. The histological score of NFIB cKO mice was significantly higher than that of non-cKO mice (4.25±0.50 vs 0.50±0.58, P＜0.01). Intestinal immunohistochemical staining showed that more CD11b positive cells were recruited in NFIB cKO mice than non-cKO mice. Conclusion Myeloid specific NFIB cKO mice have been successfully constructed, and NFIB in myeloid cells can reduce infiltration of immune cells (granulocytes or/and monocytes) to inhibit intestinal inflammation. |
| Key words: conditional gene knockout nuclear factor ⅠB intestinal inflammation myeloid cells animal models |
