论文部分内容阅读
[目的]探讨有效的肿瘤生物治疗新思路,用肝癌细胞系Bel7402-V3为细胞模型检测联合单克隆抗体9A9和15D2对其干性功能(自我更新、侵袭和耐药)的影响。[方法]采用活细胞流式免疫荧光术检测单独抗体、联合抗体与肿瘤干细胞标志物ESA在Bel7402-V3亲本及sphere细胞中的表达;甲基纤维素成球实验和Transwell小室侵袭实验分别检测抗体对Bel7402-V3亲本细胞成球能力和侵袭能力的影响;CCK8法及IC50法检测联合抗体对Bel7402-V3亲本细胞耐药能力的影响。[结果]流式荧光结果显示:标志物ESA、单抗15D2、单抗9A9所识别的细胞比例在Bel7402-V3 sphere细胞中比在亲本细胞中分别富集了2.6倍、4.1倍、2.0倍;在Bel7402-V3亲本和sphere细胞中,联合单抗与标志物共染比例(9A9+15D2与ESA)大于单抗分别与标志物共染比例之和[(9A9+ESA)+(15D2+ESA)];单抗9A9与15D2联合时对Bel7402-V3亲本细胞的成球抑制率和侵袭抑制率均明显高于等浓度的单种单抗对Bel7402-V3亲本细胞的功能抑制;经联合单抗处理的Bel7402-V3亲本细胞耐药能力较经等浓度单种单抗处理的细胞耐药能力显著降低[9A9+15D2:IC50为0.32μg/ml,9A9:IC50为0.58μg/ml,15D2:IC50为0.56μg/ml。[结论]肿瘤干细胞中存在不同亚群,其在不同的信号通路或基因水平上发挥了不同的作用。联合多靶点的抗体治疗能显著提高疗效。
[Objective] To explore a new idea of effective tumor biotherapy, and to investigate the effect of combined monoclonal antibody 9A9 and 15D2 on the dry function (self-renewal, invasion and drug resistance) of the hepatocellular carcinoma cell line Bel7402-V3 as a cell model. [Methods] The expression of ESA, a marker of ESA in Bel7402-V3 parental cells and sphere cells, was detected by flow cytometry with live cell flow cytometry. Methylcellulose scaffold and Transwell chamber invasion assay were used to detect the antibody On the ability of Bel7402-V3 parental cells to agglutinate and invasion. The effects of the combined antibodies on the drug resistance of Bel7402-V3 parental cells were detected by CCK8 assay and IC50 assay. [Results] The results of flow cytometry showed that the percentage of cells identified by ESA, mAb 9A2 and McAb 9A9 was 2.6, 4.1 and 2.0 times higher than that of Bel7402-V3 sphere cells, respectively. In the Bel7402-V3 parental and sphere cells, the combined mAb and marker ratios (9A9 + 15D2 and ESA) were greater than the sum of the mAb-specific marker co-staining ratios [(9A9 + ESA) + (15D2 + ESA) ]; Monoclonal antibody 9A9 15D2 Bel7402-V3 parental cells when combined with the ball inhibition and invasion inhibition were significantly higher than the concentration of a single monoclonal antibody Bel7402-V3 parental cell inhibition; combined mAb treatment The drug resistance of Bel7402-V3 parental cells was significantly lower than that of cells treated with the same concentration of single monoclonal antibody [9A9 + 15D2: IC50 was 0.32μg / ml, 9A9: IC50 was 0.58μg / ml, 15D2: IC50 was 0.56 μg / ml. [Conclusion] There are different subsets in cancer stem cells, which play different roles in different signal pathways or gene levels. Combined multi-target antibody treatment can significantly improve the efficacy.