靶向上皮特异性抗原阳性肝癌干细胞单克隆抗体联合顺铂治疗肝癌的实验研究  

Treatment of liver cancer in vitro and in mice by monoclonal antibody targeting epithelial specificantigen-positive liver cancer stem cells in combination with cisplatin

作　　者：何永燕 遇珑[2] 容雁[2] 孙立新[2] 孙力超[2] 杨治华[2] 冉宇靓[2] 李力[3] 

机构地区：[1]广西医科大学医学科学实验中心,南宁530021 [2]国家癌症中心中国医学科学院北京协和医学院肿瘤研究所分子肿瘤学国家重点实验室,100021 [3]广西医科大学附属肿瘤医院妇瘤科,南宁530021

出　　处：《中华肿瘤杂志》2016年第5期333-339,共7页Chinese Journal of Oncology

基　　金：国家青年科学家专题项目（863计划）（2014AA020537）;广西区域性高发肿瘤早期防治研究重点实验室开放课题（GK2013-KF01）

摘　　要：目的探讨靶向肝癌干细胞单克隆抗体的生物学特征及其联合顺铂治疗肝癌的疗效。方法采用无血清悬浮培养法和PKH26染色法确定肝癌Bel7402-V3细胞中存在肿瘤干细胞。细胞免疫荧光法检测单克隆抗体15D2识别的抗原蛋白与PKH26阳性细胞及上皮特异性抗原（ESA）在Bel7402-V3细胞中的表达情况。无血清悬浮培养法检测流式细胞术分选出的15D2阳性细胞的自我更新能力以及15D2对Bel7402．V3细胞自我更新能力的影响。CCK8法检测15D2对细胞顺铂耐药能力的影响。裸鼠体内治疗实验分析15D2联合顺铂对Bel7402．V3移植瘤生长的抑制作用。结果Bel7402-V3细胞无血清悬浮培养11d后形成的细胞球体中存在单个PKH26阳性细胞。细胞免疫荧光显示，单克隆抗体15D2识别的抗原分子能与PKH26和ESA在Bel7402-V3细胞上共定位。15D2阳性细胞和15D2阴性细胞的体外成球率分别为（30．4±3．4）％和（8．8±1．8）％，差异有统计学意义（P〈0．05）。15D2阳性细胞具有更高的顺铂耐药性，15D2阳性细胞和15D2阴性细胞的半数抑制浓度（IC50）分别为1．014和0．365μmol／L。单克隆抗体15D2能够显著抑制Bel7402．V3细胞的无血清成球，抑制率为37．5％。经15D2处理后的Bel7402-V3细胞，顺铂耐药能力明显下降，其Ic50为0．211μg／ml，而对照组的IC50为0．325μg／ml。抗体体内治疗实验结果显示，50、25、12．5mg／kg15D2组的肿瘤生长抑制率分别为82．6％、71．4％和60．0％，50mg／kg15D2＋顺铂组的肿瘤生长抑制率为91．0％，顺铂组的肿瘤生长抑制率为56．7％。结论单克隆抗体15D2是抗肝癌干细胞的功能性单克隆抗体，为肝癌干细胞靶向治疗的候选抗体药物。Objective To investigate the biological characteristics of monoclonal antibodies against human liver cancer stem cells and its therapeutic effect in combination with cisplatin in the treatment of hepatocellular carcinoma. Methods Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cells in human liver Be17402-V3 cell line. The co-expression of antigen recognized by monoclonal antibody （McAb） 15D2 and epithelial specific antigen （ESA） and PKH26-positive ceils in the Be17402-V3 ceils were detected by immunofluorescence assay. Serum-freesuspension culture was used to detect the self-renewal ability of 15D2-positive Be17402-V3 cells sorted by flow cytometry and the effect of 15D2 on the self-renewal ability of Be17402-V3 cells. The effect of 15D2 on cisplatin resistance in the cells was examined by CCK8 method. The inhibitory effect of 15D2 combined with cisplatin on the transplanted tumor growth in mice was also observed. Results Single PKH26-positive cells were observed in the Be17402-V3 cell spheroids cultured for 11 days. Immunofluoreseeuce assay showed that the 15D2-recognized antigen could be conjugated with PKH26 and ESA and co-localized on Be17402-V3 cells. The spheroid formation rate of 15D2-positive cells in serum-free medium was significantly higher than that of 15D2-negative cells [ （30.4±3.4） % vs. （ 8.8±1.8） % ,P〈0.01 ]. The cisplatin＇resistance of 15D2- positive cells was obviously higher than that of 15D2-negative cells （IC50： 1.014 μmol/L vs. 0.365 μmol/L）. MeAb 15D2 significantly suppressed the spheroid formation of Be17402-V3 cells, with an inhibition rate of 37.5%. McAb 15D2 also notably inhibited the eisplatin resistance of Be17302-V3 cells. The ICs0 was 0.211 μg/ml in the 15D2 group and 0.325 μg/ml in the control group. The mouse experiment showed that the tumor growth rates of 50 mg/kg, 25 mg/kg and 12.5 mg/kg 15D2-treatment groups were 82.6%, 71.4% and 60.0%, respectively; that of the 50 mg/kg 15D2 ±cisplatin

关 键 词：肝肿瘤 细胞系 肿瘤 肿瘤干细胞 抗体 单克隆 顺铂 上皮特异性抗原 抗药性 

分 类 号：R735.7[医药卫生—肿瘤]