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目的 :研究一株杂交瘤细胞分泌的识别上皮特异性抗原(epithelial specii c antigen,ESA)阳性肝癌干细胞的单克隆抗体(简称单抗)15B7的体外功能,为靶向肝癌干细胞的肝癌治疗提供候选抗体药物。方法 :采用无血清悬浮培养及PKH26染色法确定肝癌Bel7402-V3细胞球中存在肿瘤干细胞。采用FCM法和双色细胞免疫荧光法检测ESA和单抗15B7识别的抗原蛋白在Bel7402-V3细胞中的表达情况。采用无血清悬浮培养法观察单抗15B7作用对Bel7402-V3细胞球自我更新能力的影响,细胞计数试剂盒-8法检测单抗15B7对Bel7402-V3细胞的顺铂耐药性的影响,Transwell实验检测单抗15B7对Bel7402-V3细胞侵袭能力的影响。结果 :Bel7402-V3细胞经无血清悬浮培养11 d后形成的细胞球体中存在单个PKH26染色阳性的肿瘤干细胞。单抗15B7识别的抗原分子能与ESA一起共定位于Bel7402-V3细胞。单抗15B7能显著抑制Bel7402-V3细胞在无血清培养液中成球(P<0.05),抑制率达32.4%;单抗15B7能够有效降低Bel7402-V3细胞的顺铂耐药性(P<0.05),实验组与未经单抗15B7处理的对照组半数抑制浓度分别为0.14μg/m L和0.27μg/m L;此外,单抗15B7亦能抑制Bel7402-V3细胞的侵袭能力(P<0.05),抑制率达30.7%。结论 :杂交瘤单抗15B7在体外能够显著抑制ESA阳性肝癌干细胞的自我更新、耐药和侵袭能力,推测其可作为肝癌干细胞靶向治疗的有价值的候选抗体药物。
AIM: To investigate the in vitro function of a monoclonal antibody (mAb) 15B7 secreted by a hybridoma that recognizes epithelial specii c antigen (ESA) -positive hepatocarcinoma stem cells and to provide candidates for the treatment of hepatocellular carcinoma Antibody drugs. Methods: Tumor stem cells were determined in serum-free Bel7402-V3 cells by serum-free suspension culture and PKH26 staining. FCM and two-color cell immunofluorescence were used to detect the expression of ESA and monoclonal antibody 15B7 in Bel7402-V3 cells. The effect of mAb 15B7 on the self-renewal ability of Bel7402-V3 cells was observed by serum-free suspension culture. The effect of mAb 15B7 on the resistance of Bel7402-V3 cells to cisplatin was tested by the cell counting kit-8 method. Effect of mAb 15B7 on Invasion Ability of Bel7402-V3 Cells. RESULTS: Single PKH26 positive tumor stem cells were found in the cell spheres of Bel7402-V3 cells cultured in serum-free suspension for 11 days. Antibody molecules recognized by mAb 15B7 could co-localize with Bel7402-V3 cells with ESA. Monoclonal antibody 15B7 could significantly inhibit Bel7402-V3 cells spheroidization in serum-free medium (P <0.05) with the inhibition rate of 32.4%. McAb 15B7 could effectively reduce the cisplatin resistance of Bel7402-V3 cells (P <0.05 ). The median inhibitory concentrations (IC50) were 0.14μg / ml and 0.27μg / ml respectively in the control group and those in the control group without mAb 15B7. In addition, the monoclonal antibody 15B7 also inhibited the invasion ability of Bel7402-V3 cells (P <0.05 ), Inhibition rate of 30.7%. CONCLUSION: Hybridoma mAb 15B7 can significantly inhibit the self-renewal, drug resistance and invasiveness of ESA-positive hepatocellular carcinoma stem cells in vitro, suggesting that it may be a valuable candidate antibody for liver cancer stem cell targeted therapy.