南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (3): 428-435.doi: 10.12122/j.issn.1673-4254.2023.03.13

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雪貂食管的形态学及其分子标记物的表达谱

李 博，王 磊，赵 伟，范玉涵   

  1. 宁夏医科大学总医院胃肠外科，制剂中心，宁夏 银川 750004
  • 出版日期:2023-03-20 发布日期:2023-03-20

Morphology of the esophagus of ferrets and expression profile of molecular markers

LI Bo, WANG Lei, ZHAO Wei, FAN Yuhan   

  1. Department of Gastrointestinal Surgery, Pharmaceutical Preparation Center, General Hospital of Ningxia Medical University, Yinchuan 750004, China
  • Online:2023-03-20 Published:2023-03-20

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摘要： 目的 分析雪貂食管形态学及分子表达，探讨其作为人食管疾病动物模型的可行性。方法 取生长期为6~8月成年雪貂食管和出生1 d、3 d、5 d、1周和2周幼年雪貂食管各5只于宁夏医科大学总医院病理科制成冷冻切片、包埋石蜡组织。通过苏木精-伊红染色法（HE）染色、过碘酸雪夫染色（PAS）对成年雪貂食管粘膜下腺体（SMG）进行形态学分析；Mucin染色检测MUC5B和MUC5AC的表达情况；免疫组化、免疫荧光技术检测细胞角蛋白（CK）：CK4、CK5、CK7、CK8、CK14、CK17、CK18、CK19、CK20等相关蛋白在成年雪貂食管上皮细胞中的表达情况；免疫荧光检测LEF1在雪貂食管上皮和食管粘膜下腺中表达情况。结果 HE、PAS结果显示，成年雪貂食管粘膜下腺体为食管粘膜肌层下方结缔组织，具有分泌含有粘蛋白粘液的功能；细胞角蛋白在成年雪貂食管不同类型的细胞中呈差异性表达，粘液细胞主要表达CK4，导管细胞表达CK8，上皮细胞表达CK20，其中粘液细胞中细胞角蛋白类型表达最多；Mucin染色结果显示MUC5B、MUC5AC在成年雪貂食管腺体的粘液细胞胞质及腺体管腔内呈阳性表达；粘膜细胞中特异性结合双花扁豆凝集素（DBA）、鸡冠珊瑚树凝集素（ECL）、非偶联西非单叶豆凝集素I（GSL I) 、非偶联西非单叶豆凝集素 II（GSL II）、大豆凝集素（SBA）、他卡林生物化合物凝集素、荆豆凝集素（ULEX）、麦胚凝集素（WGA）；导管细胞细胞膜上特异性结合凝集素GSL I、GSL II，上皮细胞细胞膜凝集素ECL、花生凝集素（PNA）和WGA呈阳性表达；凝集素伴刀豆凝集素(ConA）、红腰果E型凝集素（PHA-E）和红腰果L型凝集素（PHA-L）在浆液细胞胞膜呈阳性表达。免疫荧光结果提示，随着腺体的发育，在雪貂出生后3 d至1周可见到发育腺体中LEF1的表达，当腺体分化成熟时LEF1表达消失。将第 1天至第7天龄雪貂食管腺体中LEF1表达强度与两周龄的雪貂食管腺体中荧光强度相比，差异有统计学意义（P<0.001）。结论 雪貂食管与人类食管组织具有相似的结构和部分共同标记物，尤其是与人类相似的食管粘膜下腺体，未来可将雪貂作为人食管疾病动物模型的选择之一。

关键词: 动物模型；雪貂；粘膜下腺体；形态学分析；分子表达

Abstract: Objective To examine the morphological characteristics and the expression profile of molecular markers of ferret esophagus and assess the feasibility of using ferrets as animal models for studying human esophageal diseases. Methods Frozen sections and paraffin- embedded specimens of the esophageal tissues were obtained from adult ferrets (aged 6 to 8 months) and ferrets aged 1 day, 3 days, 5 days, 1 week and 2 weeks. HE staining and periodic acid-Schiff (PAS) staining were used for morphological analysis of the esophageal submucosal glands (SMGs) of adult ferrets, and the expressions of MUC5B and MUC5AC were tested using Mucin staining; The expressions of cytokeratins (CK4, CK5, CK7, CK8, CK14, CK17, CK18, CK19, and CK20) in adult ferret esophagus were examined using HE staining and immunofluorescence assay. The expressions of LEF1 in the esophageal epithelium and SMGs were detected with immunofluorescence assay. Results In adult ferrets, the esophageal SMGs were connective tissues below the muscularis mucosa of the esophagus with secretory functions. Cytokeratins were expressed differentially in different esophageal cells: CK4, CK8 and CK20 were expressed mainly in the mucous cells, ductal cells and epithelial cells, respectively, while the mucous cells expressed the largest variety of cytokeratins. Mucin staining showed positive MUC5B and MUC5AC expression in the cytoplasm and lumen of adult ferret esophageal glands. Lectin from DBA, ECL, GSLI, GSL II, SBA, Tacalin bioylated, ULEX, WGA, GSL I and GSL II were expressed on ductal cell membrane, and ECL, PNA and WGA were detected on epithelial cell membrane. Lectin with ConA, PHA-E and PHA-L were expressed on serous cell membrane. Immunofluorescence assay showed that LEF1 in the developing glands were visible from 3 days to 1 week of age and then disappeared as the glands matured. The intensity of LEF1 expression in the esophageal glands differed significantly between ferrets aged 1 to 7 days and those aged two weeks. Conclusion Ferrets and human share similar esophageal tissue structures and some common molecular markers, suggesting the possibility of using ferrets as animal models of human esophageal diseases.

Key words: animal models; ferrets; submucosal glands; morphological analysis; molecular markers