Wang Tingting,Xu Lingqi,Gong Yuan,et al.Comparisons of animal models of Hirschsprung-associated enterocolitis[J].Journal of Clinical Pediatric Surgery,2024,(09):830-836.[doi:10.3760/cma.j.cn101785-202402017-006]
先天性巨结肠相关性小肠结肠炎动物模型造模方法的对比研究
- Title:
- Comparisons of animal models of Hirschsprung-associated enterocolitis
- Keywords:
- Hirschsprung Disease; Enterocolitis; Disease Models; Animal; Program Evaluation; Comparative Study
- 摘要:
- 目的 比较不同先天性巨结肠相关性小肠结肠炎(Hirschsprung-associated enterocolitis,HAEC)动物模型的造模方法,为HAEC发病机制的研究寻找更加可靠的动物模型。方法 将24只21日龄斯泼累格·多雷(Sprague-Dawley,SD)大鼠随机分为实验组和对照组,实验组用0.5%苯扎氯铵处理,对照组用生理盐水处理;造模3周后实验组使用大肠杆菌JM83按照每日1×109 CFU的剂量灌胃,对照组采用等量生理盐水灌胃,随后观察两组大鼠的饮食、活动及排便情况。另外选择21日龄EdnrB-/-小鼠作为实验组,同日龄野生型小鼠作为对照组,每组各5只;解剖后根据肠道大体形态将实验组小鼠肠段分为狭窄段和扩张段,采用H&E染色评估处理后肠道组织的病理变化,采用Western blot检测相关肠道屏障蛋白的表达变化。结果 成功采用苯扎氯铵包裹后大肠杆菌灌胃的方法建立 HAEC大鼠模型,成功验证21日龄EdnrB-/-小鼠作为HAEC的动物模型。两种动物模型的实验组与对照组相比,均可见活动减慢、腹部明显膨隆;解剖后可见远端肠管明显狭窄,近端肠管扩张。H&E染色发现两种动物模型的狭窄段神经节细胞团均有明显缺如,扩张段见大量炎症细胞聚集,对照组无明显异常。Western blot显示,HAEC患儿扩张肠段E-Cadherin蛋白相对表达量为0.15±0.01,低于对照组肠段的1.13±0.08(t=12.940,P＜0.001);Occludin蛋白相对表达量为0.21±0.01,低于对照组肠段的0.99±0.01(t=95.030,P＜0.001)。苯扎氯铵包裹诱导模型扩张肠段的Occludin蛋白相对表达量为0.14±0.01,低于对照组肠段的0.94±0.04(t=12.020,P＜0.001);Claudin3蛋白相对表达量为0.34±0.01,低于对照组肠段的0.99±0.01(t=38.240,P＜0.001)。EdnrB-/-小鼠模型扩张肠段的E-Cadherin蛋白相对表达量为0.28±0.01,低于对照组肠段的0.97±0.03(t=25.360,P＜0.001);Occludin蛋白相对表达量为0.32±0.01,低于对照组肠段的1.13±0.02(t=43.710, P＜0.001);Claudin3蛋白相对表达量为0.17±0.01,低于对照组肠段的1.19±0.03(t=36.960,P＜0.001)。以上差异均具有统计学意义,与临床患儿情况相符。结论 苯扎氯铵包裹后大肠杆菌灌胃的化学诱导模型及EdnrB基因敲除后的自发性动物模型都与HAEC的临床和病理特征符合,可用于HAEC病因与发病机制的研究。但EdnrB-/-小鼠作为与HD发病机制相关基因的基因敲除小鼠,不予干预而自然发生小肠结肠炎,能更好地模拟临床HAEC的病程发展和生理病理变化。
- Abstract:
- Objective To employ different modeling approaches for animal models of Hirschsprung-associated enterocolitis (HAEC) to identify a more dependable model for elucidating the pathogenesis of HAEC. Methods A total of 24 21-day-old Sprague Dawley (SD) rats were randomized into experimental group treated with 0.5% benzalkonium chloride and control group treated with saline.After 3-week modeling,experimental group was gavaged with E.coli JM83 1x109 CFU/d,and control group an equivalent amount of saline.Two groups were subsequently compared in terms of diet,activity and defecation.Additionally,21-day-old EdnrB-/- mice were selected as experimental group and wild-type littermates as control group (n=5 each).Intestinal segments of mice in experimental group were identified as narrowed and dilated segments according to gross morphology.Pathological changes were evaluated by hematoxylin-eosin (HE) stain and altered expressions of intestinal-related barrier proteins by Western blot. Results HAEC model was successfully established by gavage with E.coli after a treatment of benzalkonium chloride.And 21-day-old EdnrB-/- mice could also be utilized as an animal model of HAEC.As compared with control groups,experimental groups of both animal models showed slow activity and obvious abdominal expansion.There were obvious narrowing of distal intestine and dilatation of proximal intestine.HE stain revealed a significant absence of ganglion cell clusters in narrow segment in both animal models along with a noticeable aggregation of inflammatory cells in dilated segment.No obvious abnormality was detected in control group.Western blot analysis revealed that in HAEC patients,the relative expression levels of E-Cadherin protein in dilated segment were significantly lower than those in control group.[(0.15±0.01) vs.(1.13±0.08),t=12.940,P＜0.001]Similarly,Occludin protein expression was also lower in HAEC dilated segment than that in control group.[(0.21±0.01) vs.(0.99±0.01),t=95.030,P<0.001].In benzalkonium chloride-induced model,Occludin protein relative expression was lower in dilated segment than that in control group[(0.14±0.01) vs.(0.94±0.04),t=12.020,P＜0.001]while Claudin3 protein expression was lower than control group.[(0.34±0.01) vs.(0.99±0.01),t=38.240,P<0.001]In EdnrB-/- murine model,E-Cadherin protein expression was lower in dilated segment than that in control group[(0.28±0.01) vs.(0.97±0.03),t=25.360,P＜0.001]and Occludin protein expression was lower than control group.[(0.32±0.01) vs.(1.13±0.02),t=43.710,P<0.001]Additionally,Claudin3 protein expression was,lower than control group.(0.17±0.01) vs.(1.19±0.03),t=36.960,P<0.001].These differences were statistically significant and consistent with clinical manifestations. Conclusions The chemically induced model of E.coli JM83 gavage after benzalkonium chloride wrapping and spontaneous animal model after EdnrB gene knockout both exhibit clinical and pathological characteristics consistent with HAEC,making them suitable for elucidating the etiology and pathogenesis of HAEC.However,EdnrB-/- mice,as gene knockout mice relevant to the pathogenesis of HD,may better mimic the clinical course development and physiopathological changes of HAEC by not intervening in natural occurrence of enterocolitis.
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备注/Memo
收稿日期:2024-2-20。
基金项目:苏州市临床病种诊疗技术专项(LCZX202107);苏州市重点学科(SZXK202105)
通讯作者:黄顺根,Email:drhuang01@163.com