阪崎肠杆菌培养产物对乳鼠肠上皮细胞的损伤研究

Study on the injury of enterocyte on suckling mice induced by the culture of enterobacter Sakazakii strains

目的纯化阪崎肠杆菌（ES）培养产物,通过体内侵袭乳鼠肠上皮细胞、体外侵袭caco-2细胞,分析其对肠上皮细胞的损伤特征。方法阪崎肠杆菌用酪蛋白氨基酸液体培养基（CAYE）培养后离心取上清,硫酸铵法纯化上清并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）电泳分析。将纯化产物分别进行体内、外侵袭实验。纯化产物对3~4日龄ICR乳鼠灌胃4 d,7 d后无菌解剖取小肠,制做切片,HE染色,光镜观察;纯化产物侵袭caco-2细胞,光镜观察细胞变化。结果阪崎肠杆菌（ES）培养液上清纯化后,SDS-PAGE电泳显示纯化产物包括3个分子量相近的蛋白质;乳鼠实验表明纯化产物能够引起肠上皮细胞变性水肿、核消失;体外实验表明,纯化产物能够引起caco-2细胞形态变化并死亡。结论阪崎肠杆菌培养后能够产生具有生物活性的蛋白质,对肠上皮细胞具损伤作用。

Objective The extract from the culture of Enterobacter Sakazakii strains invaded the enterocyte and caco-2 cells of suckling mice in vivo and in vitro respectively.To analyze the characters of the injured enterocyte reduced by the extract which.Methods The supernatant which was obtained from the Culture of Enterobacter Sakazakii in casaminoacids yeast extract broth was purified by ammonium sulfate methods and analyzed by SDS-polyacrylamide gel electrophoresis.The extract invaded the enterocyte and hepatocyte in vivo by the intragastric administration in 3~4 days old ICR sulking mice for four days and the caco-2 and immature mice hepatocyte in vitro.Mice were euthanized with ethylether,and then the intestinal tract were removed for pathological section and microscopic examination after hematoxylin and eosin stain（H.E）.The extract invaded caco-2 cells and the changes of which were analyzed by the microscopic lens.Results The supernatant which were purified from the culture of Enterobacter Sakazakii strains included three different molecular weight proteins.The extract could cause the enterocyte degeneration and edema in vivo,and the caco-2 cells degeneration and death in vitro.Conclusion The Enterobacter Sakazakii could produce some proteins which possess biological activity and injure the enterocyte.