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目的探讨O-型糖链合成的抑制对肠分化细胞内细菌侵袭数量和细胞内MUC2表达水平的影响。方法对肠分化细胞(HT-29-Gal)用O-型糖链抑制剂(benzyl-N-acetyl-α-D-galactosaminide,benzyl-α-GalNAc)处理,采用Real-time PCR和Western blot检测MUC2 mRNA和蛋白的表达情况。并将HT-29-Gal细胞及benzyl-α-GalNAc处理的HT-29-Gal细胞分别与肠致病性大肠杆菌(enteropathogenic E.coli,EPEC)和肠出血性大肠杆菌(EHEC O157∶H7)37℃孵育2 h,再加入100μg/mL的庆大霉素,杀灭细胞外及粘附于细胞表面的细菌。最后采用系列稀释克隆计数法观察benzyl-α-GalNAc处理的HT-29-Gal细胞对细菌侵袭的影响。结果 Real-time PCR和Western blot检测发现经benzyl-α-GalNAc处理的HT-29-Gal细胞MUC2的mRNA和蛋白表达水平明显降低(P<0.05)。侵袭入benzyl-α-GalNAc处理的HT-29-Gal细胞的EPEC和EHEC O157∶H7的数量较对照细胞显著增加(P<0.05)。结论抑制HT-29-Gal细胞黏蛋白O-型糖链的合成导致侵袭入细胞内细菌数量增加和MUC2的表达降低。
Objective To investigate the effects of O-glycosylation on the number of bacterial invasion and the expression of intracellular MUC2 in differentiated cells of intestine. Methods The intestinal-differentiated cells (HT-29-Gal) were treated with benzyl-α-GalNAc by Real-time PCR and Western blot MUC2 mRNA and protein expression. HT-29-Gal cells and HT-29-Gal cells treated with benzyl-α-GalNAc were respectively incubated with enteropathogenic E.coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC O157: H7) After incubation at 37 ° C for 2 h, 100 μg / mL gentamycin was added to kill extracellular and bacterial cells adhering to the cell surface. Finally, the effect of benzyl-α-GalNAc-treated HT-29-Gal cells on bacterial invasion was observed by serial dilution cloning and counting. Results Real-time PCR and Western blot showed that mRNA and protein expression of MUC2 in HT-29-Gal cells treated with benzyl-α-GalNAc decreased significantly (P <0.05). The number of EPEC and EHEC O157: H7 invaded into the HT-29-Gal cells treated with benzyl-α-GalNAc was significantly higher than that of the control cells (P <0.05). Conclusion Inhibition of the synthesis of mucin O-type sugar chains in HT-29-Gal cells resulted in an increase in the number of invasive bacteria and a decrease in MUC2 expression.