抗肝癌干细胞单克隆抗体的鉴定及功能  

Identification and functional study of monoclonal antibodies against human hepatocellular carcinoma stem cells

作　　者：遇珑[1] 舒雄[2] 何永燕 孙力超[1] 杨治华[1] 冉宇靓[1] 

机构地区：[1]国家癌症中心/中国医学科学院北京协和医学院肿瘤医院细胞及分子生物学研究室,北京100021 [2]北京市创伤骨科研究所,北京100035

出　　处：《现代肿瘤医学》2017年第17期2693-2698,共6页Journal of Modern Oncology

基　　金：国家自然科学基金(编号:31371445)

摘　　要：目的:鉴定可靶向肝癌干细胞的功能性单克隆抗体,为肝癌干细胞靶向治疗提供抗体候选药物。方法:以肝癌细胞系Bel7402-V3为模型,采用流式细胞术分选ESA^+细胞后检测其耐药能力及致瘤能力。双色细胞免疫荧光检测单抗3G7和ESA识别的抗原蛋白在Bel7402-V3细胞中的表达情况,同时采用此法检测sphere中PKH26与3G7的共染情况。流式细胞术分选3G7^+细胞后检测其自我更新能力,并采用CCK-8法检测其耐药能力;甲基纤维素成球实验,检测单抗3G7对细胞自我更新能力的影响;CCK-8法检测单抗3G7对细胞增殖和耐药能力的影响。结果:流式细胞术分选ESA^+细胞的耐药性明显高于ESA-细胞,其IC_(50)值分别为2.30μmol/L、0.49μmol/L(P<0.01),ESA^+细胞的致瘤性较ESA-细胞高至少40倍。细胞免疫荧光结果显示单抗3G7识别的抗原分子能与ESA在Bel7402-V3细胞上共定位,并能与标识干细胞的PKH26染料共染。流式细胞术分选3G7^+细胞体外成球率明显高于3G7^-细胞[(30.4±3.4)%vs(8.8±1.8)%](P<0.01),耐药性也明显较高(IC_(50)值:1.014μmol/L vs 0.365μmol/L)。抗体体外功能研究发现,单抗3G7能显著抑制Bel7402-V3的甲基纤维素成球,抑制率达到37.2%;同时,单抗3G7能抑制sphere细胞的增殖,抑制率为41.7%(P<0.01);经单抗3G7处理过细胞的耐药能力显著降低,实验组与对照组的IC_(50)分别为0.56μg/ml和0.68μg/ml。结论:单克隆抗体3G7是一株抗肝癌干细胞的功能性单抗,为肝癌干细胞靶向治疗的候选抗体药物。Objective： To screen and identify functional monoclonal antibodies （ McAbs） against human liver cancer stem cells （CSCs） ,to provide candidate antibody drugs for the stem cell - targeted therapy of liver cancer. Methods：Taking hepatocellular carcinoma cell line Bel7402 - V3 as a cell model,the expression of hybridoma mono-clonal antibody 3G7 in hepatocellular carcinoma parent cells and sphere cells was studied by flow cytometry. The ex-pression of antigen protein, which was identified by both monoclonal antibody 3G7 and ESA in Bel7402 - V3 cell, was detected by two - color immunofluorescence. Meanwhile, two - color immunofluorescence was used to detect the co - dyeing of PKH26 and 3G7 in Bel7402 - V3 sphere cells. Sphere colony formation assay was used to the self - renewal capability of 3G7 ＋ cells sorted by flow cytometry, and the resistance ability of 3G7＋ cells was examined by CCK -8 assay. Sphere colony formation assay and CCK - 8 assay were respectively used to detect the effect of monoclonal anti-body 3G7 on the self - renewal capability, cell proliferation and resistance ability of Bel7402 - V3 cells. Results ： The expression of 3G7 in Bel7402 - V3 sphere cells was 2. 8 fold than Bel7402 - V3 parent cells. The cell immunofluores-cence analysis showed that the antigen identified by 3G7 and ESA could be co - localized in Bel7402 - V3 cells,and the antigen identified by 3G7 was co - stained with PKH26 dye. Sphere formation abilities in 3G7 ＋ cells and 3 G7 Bel7402 -V3 cel lswereident ified[（30.4±3.4）%i vs （8.8±1.8）%].The chemothempy resistance of3G7＋ cells was significantly higher than that of 3 G7 cells （ IC50 ： 1.014 jjimol/L vs 0. 365 jjimol/L） . The study on functional properties of antibody in vitro and in vivo illustrated that： 3G7 mAb could significantly inhibit sphere formation of Bel7402 - V3 cells and the inhibition rate reached （ 26. 9 ± 1 2 ） % , 3G7 mAb could inhibit the proliferation ofsphere cells,and the inhibition rate wa

关 键 词：肝癌干细胞 单克隆抗体 成球 耐药 

分 类 号：R73-36[医药卫生—肿瘤] R735.7[医药卫生—临床医学]