中华眼镜蛇细胞毒素及其偶联膀胱癌单克隆抗体的体外抑制膀胱癌细胞增殖的实验研究  

In vitro cytotoxicity following specific activation of a bladder cancer-associated monoclonal antibody conjugated to a cytotoxin from Naja Naja Atra.

作　　者：王剑松[1] 丁明霞[1] 李翀[2] 谢蜀生[2] 王婉瑜[3] 徐鸿毅[1] 

机构地区：[1]昆明医学院第二附属医院泌尿外科,云南省泌尿外科研究室,昆明650101 [2]北京大学医学部基础医学院免疫系,北京100083 [3]中国科学院昆明动物所,昆明650223

出　　处：《云南医药》2007年第1期1-5,共5页Medicine and Pharmacy of Yunnan

基　　金：云南省科技攻关项目(2002NG18)

摘　　要：目的从中华眼镜蛇的四种细胞毒素(cytotoxins,CTXs)中筛选活性最强的一种与膀胱癌单克隆抗体构建成免疫毒素(immunotoxin IT),并研究IT的体外抑制膀胱癌细胞增殖的活性。方法[3H]-TdR掺入法检测CTXs对膀胱癌细胞株的抑制增殖作用,用双功能偶联剂将CTX-12与BDI-1连接成IT,并用SDS-聚丙烯酰胺凝胶电泳、DAB染色法和[3H]-TdR掺入法鉴定和检测IT的活性。结果四种CTXs对BIU-87和EJ细胞株均有抑制增殖的活性,CTX-12与BDI-1能够连接成IT,IT对BIU-87和EJ细胞株具有特异性结合性。且IT仍具有抑制肿瘤细胞增殖的活性,其BIU-87,EJ和Lovo细胞作用6h的IC50分别为97.12,85.28和193.4mg/L,而作用半小时更换培养液培养至6h的IC50分别为94.64,81.28和865mg/L。结论四种CTXs对膀胱癌细胞株均有抑制增殖的活性,CTX-12与BDI-1连接的IT具有特异性抑制增殖的活性,IT和四种CTXs都有潜力成为新的抗膀胱癌药物。Objective The inhibiting tumor cell proliferate actives of four cytotoxins （CTX-10, CTX-11, CTX-12 and CTX13） were tested using two human bladder tumor cell lines （BIU-87 and EJ） . The CTX-12 which owned the strongest activity was chosen and the immunotoxin （IT） was prepared by the CTX-12 conjugated to a bladder cancer-associated monoclonal antibody （BDI-1） The antitumor actives of IT on BIU-87 and EJ cell lines were tested. Methods The inhibiting tumor cell proliferate actives of four cytotoxins were tested by [^3H]-TdR incubated analysis. CTX-12 and BDI-1 were covalent coupled by chemical methods with heterobifunctional cross-linking reagents （SPDP） The disulfide-linked BDI-1-CTX-12 complexes were detected by SDS-gradient polyacry- lamide gel electrophoresis. Using DAB dyeing to analyze the IT binding to BIU-87 and EJ cell lines and using [^3H] -TdR incubated analysis to analyze the IT＇s inhibiting tumor cell proliferate active. Results All of the four CTXs showed favorable inhibiting bladder tumor cell proliferate actives and the inhibitory effects showed a definite dose-effect relationship. The IC50 （6h incubation） of CTX-10, CTX-11, CTX-12 and CTX13 on BIU-87 was 6.03, 6.27, 2.25 and 2.89mg/L, the IC50 （6h incubation） on EJ was 8.02, 7.89, 3.05 and 2.76 mg/L respectively. SDS-PAGE demonstrated that CTX-12 and BDI-1 could be conjugated. The IT owned a specific binding to BIU-87 and EJ cell lines, whereas it could not bind to colonic carcinoma cell lines （Lovo） The IT could inhibite proliferation of malignant tumor cell lines. The IC50 （6h incubation） of it on BIU-87, EJ and Lovo cell lines was 97.12, 85.28 and 193.4 mg/ L respectively. Whereas, if the IT replaced by RPMI1640 after incubated 30 min and continued incubating to 6h, the IC50 on BIU-87, EJ and Lovo cell lines was 94.64, 81.28 and 865 mg / L respectively. Conclusion Four CTXs own favorable inhibiting bladder tumor cell proliferate actives. The IT prepared by conjugating CTX-12 with BDI-1 shows selective cytotox

关 键 词：细胞毒素 膀胱癌 免疫毒素 增殖 

分 类 号：R996.3[医药卫生—毒理学]