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

胃粘液被认为可以保护胃黏膜免受机械损伤、干燥、病原微生物、酸和蛋白酶的侵害。采用含胍缓冲液增溶、凝胶渗透层析、蓖麻凝集素(RCA)- i亲和层析、GSA- ii亲和层析等方法，从大鼠胃黏膜中分离纯化单纯蓖麻凝集素(GSA)- ii结合黏液糖蛋白（GMG）。大鼠GMG在Sephacryl S-1000色谱柱上显示高分子量，在0.5%琼脂糖-2%聚丙烯酰胺复合凝胶和印迹中显示单带。GMG制剂中含有约60kDa的蛋白质。用三氟甲烷磺酸处理GMG去糖基化。培养抗去糖基化GMG（deGMG）的抗体。该抗体识别deGMG、GMG、周期性酸处理的deGMG和o -甘聚糖酶消化的dGMG，但对胰蛋白酶消化的deGMG没有反应。这些结果表明抗体识别GMG的肽主链。免疫组化法对胃腔表面粘膜凝胶层进行抗体染色。该抗体不仅能识别胃粘膜颈细胞和幽门腺细胞，还能识别胃表面黏液细胞、十二指肠腺黏液细胞、小肠和结肠杯状细胞。说明GMG是大鼠胃粘液的组成成分，该抗体可识别大鼠胃和肠内的粘液分泌细胞。

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.