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IC50值分别为2.30 μmol/L、0.49 μmol/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(IC50值:1.014 μmol/L vs 0.365 μmol/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IC50分别为0.56 μg/ml和0.68 μg/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 monoclonal antibody 3G7 in hepatocellular carcinoma parent cells and sphere cells was studied by flow cytometry.The expression 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 antibody 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 immunofluorescence 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 3G7-Bel7402-V3 cells were identified[(30.4±3.4)% vs (8.8±1.8)%].The chemotherapy resistance of 3G7+ cells was significantly higher than that of 3G7-cells (IC50:1.014 μmol/L vs 0.365 μmol/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 of sphere cells,and the inhibition rate was 41.7% (P<0.01).The chemotherapy resistance of cells treated by 3G7 mAb was significantly reduced,and IC50 of the control group and experimental group were respectively 0.56 μg/ml and 0.68 μg/ml.Conclusion:3G7 mAb is one of functional monoclonal antibodies targeting liver cancer stem cells,which could be a candidate drug for the targeted therapy of liver stem cell.
