Immunohistochemical and molecular biological study on GSA-II binding epithelial cells of alimentary tract

胃粘液被认为保护胃粘膜免受机械创伤、干燥、病原微生物、酸和蛋白酶的伤害。我们从大鼠胃粘膜中纯化了Griffonia simplicifolia凝集素（GSA）-II结合粘液糖蛋白（GMG），通过在含胍缓冲液中溶解，凝胶渗透色谱，蓖麻凝集素（RCA）-I-亲和色谱和GSA-II-亲和色谱。大鼠GMG在Sephacryl S-1000柱上显示高分子量，在0.5%琼脂糖-2%聚丙烯酰胺复合凝胶和印迹中显示单一条带。GMG制剂中含有约60 kDa的蛋白质。GMG用三氟甲磺酸处理去糖基化。制备了抗去糖基化GMG（deGMG）的抗体。该抗体能识别deGMG、GMG、过碘酸处理的deGMG和O-聚糖酶消化的dGMG，而不与胰蛋白酶消化的deGMG反应，表明该抗体能识别GMG的肽骨架。该抗体不仅识别胃粘液颈细胞和幽门腺细胞，而且识别胃表面粘液细胞、十二指肠腺中的粘液细胞以及小肠和结肠中的杯状细胞。这表明GMG是大鼠胃粘液的组分，并且抗体识别大鼠胃和肠中的粘液分泌细胞。

Gastric mucus is thought to protect the gastric mucosa from mechanical trauma, desiccation, pathogenic microorganisms, acid and proteases. We purified Griffonia simplicifolia agglutinin (GSA)-II-binding mucus glycoprotein(GMG)from rat gastric mucosa by solubilization in a guanidine-containing buffer, gel permeation chromatography, Ricinus communis agglutinin(RCA)-I-affinity chromatography and GSA-II-affinity chromatography. Rat GMG showed high molecular weight on a Sephacryl S-1000 column, and a single band in 0.5%agarose-2% polyacrylamide composite gels and blots. A protein of about 60kDa was contained in the GMG preparation. GMG was deglycosylated with trifluoromethanesulfonic acid treatment. An antibody was rraised against deglycosylated GMG(deGMG). The antibody recognized deGMG, GMG, periodic acid-treated deGMG, and O-glycanase-digested dGMG, but did not react to trypsin-digested deGMG.These results that the antibody recognizes the peptide backbone of GMG.In imminohistochemistry, the mucous gel layr of the stomach luminal surface was stained with the antibody. The antibody recognized not only gastric mucous neck cells and pyloric gland cells but also gastric surface mucous cells, mucous cells in the duodenal gland, and goblet cells in the small intestine and colon. these indicate that GMG is a component of rat gastric mucus, and that the antibody recignizes mucous-secreting cells in rat stomach and intestine.