胆管癌胆管胆汁中糖蛋白糖链结构变化及其酶学机制

Primary Studies on the Changes of Structures of Sugar Chains and the Mechanisms of N-acetylglucosaminyltransferase(GnT) in Cholangiocarcinoma Bile

目的：研究胆管癌胆汁中糖蛋白糖链结构的变化及其酶学机制,探讨胆汁中糖蛋白成份及其糖链结构的改变在良恶性胆道病变中的鉴别诊断价值。方法：运用各种生化技术将一组恶性胆道病变(胆管癌27例、胆囊癌9例、壶腹癌5例)和20例良性胆道病变所致梗阻性黄疸病人胆管胆汁中的蛋白分离出来,进行糖蛋白定性。采用制备好的HRP标记凝集素探针(DSA-HRP、WGA-HRP、LCA-HRP、ConA-HRP)分别对良恶性胆管胆汁中的糖蛋白的糖链结构进行检测和分析。将已分离、纯化和鉴定好的GnT-Ⅲ、Ⅳ、Ⅴ的最适受体底物与上述胆管癌、胆囊癌组织及良性胆管组织的酶液进行反应,再以HPLC测定组织中GnT-Ⅲ、Ⅳ、Ⅴ的活力。结果：良恶性胆道病变的胆汁中糖蛋白的糖链结构存在明显差异,胆管癌胆汁中糖蛋白的糖链结构的变化主要表现为天线数和核心岩藻糖的明显增多,还可能伴有平分型GlcNAc的增多。GnT-Ⅴ、Ⅳ、Ⅲ的活力在胆管癌和胆囊癌癌组织中明显高于良性对照组,尤以GnT-Ⅴ、Ⅲ活力增高更明显。结论：胆管癌胆汁中的糖蛋白及其糖链结构的变化可能对胆管癌的定性诊断有一定价值。胆管癌组织中糖基转移酶活力的改变是胆汁中糖蛋白糖链结构变化的酶学机制。

Objective：In this study, the changes of glycoproteins components and the structures of their sugar chains and the mechanisms of N-GnT in cholangiocarcinorna bile were primarily studied, in order to find a valuable index in the diagnosis of cholangioearcinorna. Methods：By using several biochemical methods, the component of proteins in bile of 41 patients with malignant biliary diseases （27 cases of eholanglocarcinorna, 9 cases of gallbladder cancer, 5 eases of cancer of vater＇s） and 20 patients with benign biliary diseases, were studied. By means of staining the glycoprotein by a kit from Bio-Rad company, it can be characterized if the proteins in bile were glycoprotein or not. The structures of the sugar chains in glyproteins from both malignant and benign biliary diseases bile were analyzed by the probes of lectin-HRP（DSA-HRP, WGA-HRP, LCA HRP and ConA HRP）. The activities of N-GnT Ⅲ, GnT Ⅳ and GnT Ⅴ from tissue of hilar cholangiocarcinorna, gallblaclder cancer and normal tissue were further determined by using high performance liquid chromatography（HPLC） with a C18 colum in the presence of prepared flurescence labeled sugar chain, GlcNAc2Man3GIcNAc2-PA. Results： There were both amounts and structural changes of glycoprotein in cholangiocarcinorna and gallblaclder cancer bile. The increase of antennary and core-fucose were the main changes in the sugar chains of these glycoproteins in cholangiocarcinorna bile, and maybe accompanied with the increase of Bisecting-GleNAc. It was found that the activities of GnT Ⅲ, GnT Ⅳ and GnT Ⅴ（especially GnT-Ⅴ, Ⅲ） both from hilar cholangiocarcinorna and gaUblaclder cancer were much higher than those from normal tissues. Conclusion： All of the results suggested that the changes of both contents and structures of glycoprotein in cholangiocarcinoma bile become a valuable index for the differential diagnosis of cholangiocarcinoma. The mechanisms of these changes were the results of the increasement of GnT activity in membrane of cell of cholangiocarcinorna and gallblaclder tissues.