O型糖链合成阻滞对肠上皮细胞MUC2表达及细菌黏附的抑制作用

Inhibitory effect of O-glycosylation inhibition on human intestinal epithelial cells Mucin 2 expression and bacteria adherence

目的探讨肠上皮细胞O型糖链的合成阻滞对该细胞肠分化标记物MUC2表达及细菌黏附的影响。方法采用O型糖链抑制剂benzyl-α-GalNAc抑制结肠上皮细胞HT-29及其分化型细胞(HT-29-Gal)O型糖链的合成,经benzyl-α-GalNAc处理的HT-29和HT-29-Gal细胞分别命名为HT-29-OBN和HT-29-Gal-OBN。采用Real-time PCR和Western blotting方法检测上述4种细胞中MUC2基因的转录和蛋白表达水平,并将上述细胞与致病性大肠埃希菌(EPEC)和肠出血性大肠埃希菌(EHEC)O157:H7共培养,采用系列稀释菌落计数法观察细菌在上述细胞表面的黏附情况。结果 Realtime PCR和Western blotting结果显示,经benzyl-α-GalNAc处理后,HT-29和HT-29-Gal细胞MUC2的mRNA和蛋白表达均明显减少(P<0.05)。HT-29-OBN和HT-29-Gal-OBN细胞与致病性大肠埃希菌EPEC和EHEC O157:H7的黏附明显少于HT-29和HT-29-Gal细胞(P<0.05)。结论抑制肠上皮细胞O型糖链的合成可阻碍细菌黏附及MUC2的表达。

Objective To investigate the effect of O-glycosylation inhibition in intestinal epithelial cells on the expression of Mucin 2 （MUC2） and bacterial adherence. Methods Intestinal epithelial cells HT-29 and differentiated HT-29 cells （HT-29- Gal） were treated with an inhibitor of O-glycosylation （benzyl- α -GalNAc）, and then named as HT-29-OBN and HT-29-Gal-OBN, respectively. The mRNA and protein expression of MUC2 in HT-29, HT29-Gal, HT-29-OBN and HT-29-Gal-OBN were detected by real-time PCR and Western blotting. Then the four kinds of above cells were incubated with enteropathogenic Escherichia coli （EPEC） or enterohemorrhagic Eschcrichia coli serotype O157：H7 （EHEC O157：HT）. The bacteria were quantified by determining the colony forming unit （CFU） following the plating of serial dilutions of the bacteria to evaluate the effect of benzyl-α -GalNAc on bacteria adherence. Results The results of real-time PCR and Western blotting showed that the mRNA and protein expression levels of MUC2 in HT-29-OBN and HT-29-Gal-OBN cells were significantly lower than those in the untreated cells HT-29 and HT- 29-Gal （P〈0.05）. The bacterial adherence assay showed that the adherence of EPEC and EHEC O157：H7 to HT-29-OBN and HT- 29-Gal-OBN cells significantly decreased compared with that to HT-29 and HT-29-Gal cells （P〈0.05）. Conclusion Inhibition of O-glycosylation in intestinal epithelial cells may reduce the bacteria adherence and MUC2 expression.