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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 To purify E. coli from Enterobacter sakazakii (ES) and inoculate invasive rat intestinal epithelial cells in vitro to infect caco-2 cells in vitro. The injury characteristics of intestinal epithelial cells were analyzed. Methods Enterobacter sakazakii was cultured in CAYE medium and centrifuged. The supernatant was purified by ammonium sulfate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The purified products were in vitro and in vivo invasion experiments. The purified product was infused into ICR neonatal rats aged 3-4 days for 4 days. After 7 days, the small intestine was aseptically dissected and made into sections and stained with HE and observed with light microscope. The purified product was used to invade caco-2 cells and observed by light microscopy. Results After purification of E. sakazakii (ES) culture supernatant, SDS-PAGE electrophoresis showed that the purified product consisted of three proteins of similar molecular weight. The results of sucking mice showed that the purified product could cause degeneration and edema of intestinal epithelial cells. , The purified product can cause caco-2 cell morphological changes and death. Conclusion Enterobacter sakazakii can produce biologically active proteins and cause damage to intestinal epithelial cells.