膀胱癌EJ细胞的CD44异常糖基化及其单抗KMP1的生物学功能研究

Aberrant Glycosylation of CD44 in Oncogenesis of EJ Cells and Biological Effects of Anti-CD44 Monoclonal Antibody KMP1

目的:研究EJ细胞单抗KMPl的结合抗原CD44的变化和KMPl对EJ细胞生物学功能的影响。方法:采用人膀胱癌EJ细胞株免疫BALB/c小鼠,获得单抗KMPl,免疫荧光检测KMPl的亲和性、结合部位及效价。流式细胞检测KMPl对膀胱癌细胞的特异性。免疫组化检测对膀胱癌组织的特异性。亲和层析分离纯化特异性抗原,检测抗原的氨基酸序列。糖基转化酶抑制剂和过碘酸氧化后分析抗原决定簇的理化性质改变。软琼脂培养克隆形成实验和细胞划痕实验检测KMPl对EJ细胞株的增殖和迁移功能的影响。结果:获得的单抗KMPl是IgGl,能与EJ、BIU-87、T24膀胱癌细胞和膀胱癌组织特异性结合,而与Lovo、HeLa、K562、HepC2、Jurkat、293、HCV29、人的红细胞和白细胞无结合性,也不能结合正常膀胱黏膜。其结合EJ细胞的抗原是异常糖基化的CD44,糖基转化酶抑制剂BC作用EJ细胞7d,KMPl对EJ细胞结合性降低,过碘酸氧化后Western blot显示KMPl对CD44结合性降低,软琼脂培养克隆形成实验和细胞划痕实验结果示KMPl还能减弱EJ细胞的增殖和迁移功能。结论:膀胱癌的高复发性、易转移可能与出现异常糖基化的CD44高表达具有一定的相关性,KMPl可结合异常糖基化的CD44,并抑制EJ细胞的增殖和迁移。

Objective： To examine the variation in the antigen CD44 when immuno-conjugated with the monoclonal antibody KMP1 and the biological effects of KMP1 on EJ cells. Methods： KMP1, the antibody against bladder cancer that could recognize the aberrantly glycosylated CD44 of human EJ cells, was obtained from BALB/c mice. ELISA was used to determine the affinity, binding site, and valence of this antibody. Flow cytometry and immunohistochemistry were conducted to detect the specificity of this antigen on the EJ cells and cancer tissues. The specific antigen was separated and purified by affinity chromatography, and its amino acid sequence was determined. After glycosyltransferase inhibition by benzyl-N-acetyl-d-galactosaminide （ BG ） and mild periodate oxidation, the variation in the physico-chemical property of the antigenic determinant was analyzed. Soft agar clone formation and wound healing as- says were performed to investigate the effect of KMP 1 on the proliferation and migration of the EJ cells. Results： KMP1 was an IgG1 antibody that can be specifically bound to the E J, BIU-87, and T24 cell lines as well as to bladder carcinoma tissue, but not to the Lovo, HeLa, K562, HepG2, Jurkat, 293, HCV29 cell lines, the human red blood cell, human lymphocytes, or normal bladder tissue. KMP1 recognizes aberrantly glycosylated CD44 of EJ cells. BG inhibited the conjugation of KMP1 and EJ cells. KMP1 inhibits cancerous proliferation and migration of the EJ cells. Conclusions： The high recurrence and metastasis of the bladder carcinoma may be in a definite relation to the high expression of CD44 with aberrant glycosylation. KMP1 can bind the aberrantly glycosylated CD44 and inhibit the proliferation and migration of the EJ cells.