唾液酸粘蛋白单抗5E2、6C10的制备、鉴定及对胃肠癌糖链表达规律的初步探讨  

Preparation and Characterization of Sialyl - mucin Specific Monoclonal Antibodies and their Immunohistochanical Usage in Gastric Cancer and Colorectal Carcinoma

作　　者：靳蕾[1] 王润田[1] 张页[2] 李津辉 姚卫东[4] 

机构地区：[1]北京大学医学部公卫学院流行病学与卫生统计学系,100083 [2]北京大学医学部基础医学院细胞生物学系 [3]山东省烟台市牟平医院病理科 [4]山东省烟台市毓璜顶医院病理科

出　　处：《中国预防医学杂志》2002年第1期18-23,共6页Chinese Preventive Medicine

摘　　要：目的 寻找识别新的肿瘤相关粘蛋白抗原的单抗,为阐明胃肠癌粘液糖蛋白的表达规律进而为胃肠肿瘤的预后、分型等提供参考依据。方法 以牛颌下腺粘蛋白(BSM)为免疫原免疫小鼠,以免疫脾细胞与Sp2/0细胞融合建立杂交瘤细胞株,用有限稀释法克隆化,间接ELISA及间接ELISA结合神经氨酸酶消化筛选克隆。以聚丙烯酰胺凝胶电泳,免疫印记(Western-Blot),间接ELISA结合热处理、胰蛋白酶消化、过碘酸氧化以及稀碱处理去除O-乙酰基等方法进行抗体的鉴定。以免疫组化LSAB法观察两株抗体对68例胃癌和55例肠癌粘蛋白糖链抗原的标记情况。结果 两株抗体分别命名为5E2和6C10,Western-Blot显示两株抗体均只与分子量大于220kDa的PAS染色阳性条带反应。两株单抗与BSM的反应都对热处理、胰蛋白酶消化、过碘酸氧化、神经氨酸酶消化等处理敏感。5E2还对稀碱去除O-乙酰基敏感,6C10可以部分吸收sialyl-lewis^a。单抗6C10在早期胃癌及癌旁的阳性率分别为5.5％和52.9％,进展癌及其癌旁的阳性率分别为14.0％和22.9％;在结肠癌及其癌旁的阳性率分别为30.9％和83.8％。单抗5E2在胃早期癌及其癌旁的阳性率分别为16.7％和41.2％,在进展癌及其癌旁的阳性率分别为42.0％和37.5％;在肠癌及其癌旁的阳性率分别为69.1％和91.9％。结论Objective Preparing new monoclonal antibody to define the tumor associated sialyl- mucins, to find out new avenues and elucidate the expression characteristics of gastric and colonic mucins and it s usage in study of natural history of intestinal gastric cancer and colon adenocarcinomas.Methods Mice were immunized with bovine submaxillary mucin (BSM), the splenic lymphocytes from the immunized mouse were fused with the mouse myeloma Sp2/0 cells using PEG.The hybridoma cultural supernatant was screened by indirect ELISA and neuraminidase digestion combined indirect ELISA. LSAB immunohistochemical stains were performed on 123 cases formalin fixed, paraffin wax embedded human tissues, 68 of them were gastric cancer, and 55 of them were colorectal carcinoma. Results The two monoclonal antibodies raised against the sialyl - parts of BSM, named 5E2 and 6C10.Their reaction with BSM were blocked by mild KOH, heating, trypsin, periodicacid and neuraminidase treatment.5.5% early GC and 52.9% gastric mucosa adjacent to early GC; 14% advanced GC, and 22.9% gastric mucosa adjacent to advanced GC; 30.9% colon crcinoma while 83.8% colon mucosal adjacent to the cancer, were stained by McAb6C10.16.7% early GC and 41.2% gastric mucosa adjacent to early GC; 42.0% advanced GC, and 37.5% gastric mucosa adjacent to advanced GC; 69.1 % colon carcinoma and 91.9% colon mucosa adjacent to the cancer were stained by McAb5E2.Conclusion: The epitopes of McAb5E2 was showed constructed by Sia Modified by 0-acetyl group but the epitop of McAb6C10 only correlated with Sia.McAb6C10 maybe useful in study of expression of mucins in nature history of intestinal GC and its application, and McAb5E2 may be a useful tool in study of distribution of O - acetylated sialyl - mucins and its application.

关 键 词：胃肿瘤 肠肿瘤 唾液酸粘蛋白 单克隆抗体 5E2 6C10 糖链 蛋白表达 

分 类 号：R735[医药卫生—肿瘤]