抗胃癌干细胞单克隆抗体的筛选鉴定及功能研究

Screening,identification and functional study of monoclonal antibodies against gastric cancer stem cells

目的:筛选鉴定靶向胃癌干细胞的功能性单克隆抗体,为胃癌干细胞靶向治疗提供抗体候选药物。方法:以胃癌细胞系BGC-823为模型,采用流式细胞术分选CD44+细胞后检测其自我更新和致瘤能力。双色细胞免疫荧光检测单抗11H5和CD44在BGC-823细胞中的表达情况。流式细胞术分选11H5+细胞后检测其自我更新能力、细胞增殖和耐药能力。结果:流式细胞术分选CD44+细胞成球率明显高于CD44-细胞[(27.2±1.6)%vs(12.9±1.2)%](P<0.01),CD44+细胞的致瘤性比CD44-细胞至少高100倍。细胞免疫荧光结果显示单抗11H5识别的抗原能与CD44在BGC-823细胞上共定位。流式细胞术分选11H5+细胞体外成球率明显高于11H5-细胞[(21.4±2.0)%vs(6.2±1.0)%](P<0.01),耐药性也明显高于11H5-细胞(IC50值:0.986μmol/L vs 0.315μmol/L)。抗体体外功能研究发现,单抗11H5能显著抑制BGC-823细胞成球,抑制率达到47.9%。单抗11H5能抑制其sphere细胞的增殖,抑制率为44.9%(P<0.05)。经单抗11H5作用的耐药能力显著降低(IC50值:0.52μg/ml vs 0.64μg/ml)。结论:单克隆抗体11H5是一株抗胃癌干细胞的功能性单抗,为胃癌干细胞靶向治疗的候选抗体药物。

Objective：To screen and identify functional monoclonal antibodies （mAbs） against human gastric cancer stem cells （CSCs），and to provide candidate antibody drugs for the stem cell-targeted therapy of gastric cancer.Methods：Taking gastric cancer cell line BGC-823 as a cell model，CD44＋ cells were sorted by flow cytometry，identified the self-renewal and tumorigenicity capability.The expression of antigen protein，which was identified by both monoclonal antibody 11H5 and CD44 in BGC-823 cell，was detected by two-color immunofluorescence.Meanwhile，sphere colony formation assay and CCK-8 assay were used to detect the effect of monoclonal antibody 11H5 on the self-renewal capability，cell proliferation and resistance ability of BGC-823 cells.Results：The sphere colony rate of CD44＋ cells sorted by flow cytometry was significantly higher than that of CD44-cells ［（27.2±1.6）% vs （12.9±1.2）%］.The cell immunofluorescence analysis showed that the antigen identified by 11H5 and CD44 could be co-localized in BGC-823 cells.Sphere formation abilities in 11H5＋ cells and 11H5-cells were identified ［（21.4±2.0）% vs （6.2±1.0）%］.The chemotherapy resistance of 11H5＋ cells was significantly higher than that of 11H5-cells （IC50：0.986 μmol/L vs 0.315 μmol/L）.11H5 mAb could significantly inhibit sphere formation of BGC-823 cells，and the inhibition rate reached 47.9%.11H5 mAb could inhibit the proliferation of sphere cells，and the inhibition rate was 44.9% （P〈0.05）.The chemotherapy resistance of cells treated by 11H5 mAb was significantly reduced （IC50 value：0.52 μg/ml vs 0.64 μg/ml）.Conclusion：11H5 mAb is one of functional monoclonal antibodies targeting gastric cancer stem cells，which could be a candidate drug for the targeted therapy of gastric cancer stem cells.