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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 study the changes of the binding antigen CD44 of EJ monoclonal antibody KMP1 and the effect of KMP1 on the biological function of EJ cells. Methods: BALB / c mice were immunized with human bladder cancer cell line EJ to obtain monoclonal antibody KMP1. The affinity, binding site and potency of KMP1 were detected by immunofluorescence. Flow cytometry was used to detect the specificity of KMP1 in bladder cancer cells. Immunohistochemical detection of bladder cancer tissue specificity. Affinity chromatography separation and purification of specific antigens, detection of antigen amino acid sequence. Glycosyltransferase Inhibitors and Periodic Acid Oxidation The physicochemical properties of the antigenic determinants were analyzed. The soft agar culture clone formation assay and cell scratch assay were used to detect the effect of KMP1 on the proliferation and migration of EJ cell line. Results: The obtained monoclonal antibody KMP1 was IgG1 and could specifically bind to EJ, BIU-87, T24 bladder cancer cells and bladder cancer tissues, but not to Lovo, HeLa, K562, HepC2, Jurkat, 293, HCV29, Leukocytes without binding, it can not be combined with normal bladder mucosa. The binding of EJ cell antigen is abnormally glycosylated CD44, glycosyltransferase inhibitor BC role of EJ cells 7d, KMPl on EJ cell adhesion decreased, after periodate oxidation Western blot showed that KMPl decreased CD44 binding, soft The result of agar culture colony formation experiment and cell scratch test showed that KMP1 can also weaken the proliferation and migration of EJ cells. Conclusion: The high recurrence and metastasis of bladder cancer may be related to the high expression of abnormally glycosylated CD44. KMP1 can bind to abnormally glycosylated CD44 and inhibit the proliferation and migration of EJ cells.