抗Trop-2 IgG抗体联合顺铂对卵巢癌细胞特性的影响

Effect of anti-Trop-2 antibody IgG combined with cisplatin on the characteristics of ovarian cancer cells

目的探讨抗Trop-2 IgG抗体联合顺铂对卵巢癌上皮细胞的影响。方法转染Trop-2质粒至293F细胞,利用Protein A亲和柱纯化,获得人源抗Trop-2 IgG抗体。酶联免疫吸附试验(ELISA)检测Trop-2质粒转染效率;聚丙烯酰胺(SDS-PAGE)鉴定抗Trop-2 IgG抗体纯化效率;分子互作Biacore T100检测抗Trop-2 IgG抗体与Trop-2抗原的亲和力。蛋白免疫印迹、流式细胞术和免疫荧光检测HO8910细胞和Iose 386细胞中Trop-2蛋白表达水平。实验分为4组:磷酸盐缓冲液(PBS)组、Trop-2 IgG组、cisplatin组、Trop-2 IgG+cisplatin组。CCK8检测卵巢癌细胞存活率,Transwell检测卵巢癌细胞迁移和侵袭能力。结果成功转染并纯化人源抗Trop-2 IgG抗体,并且其与Trop-2具有较高的结合力和免疫活性。抗Trop-2 IgG抗体在HO8910细胞中高表达,在Iose 386细胞中不表达。当顺铂存在时,Trop-2蛋白表达水平在HO8910细胞中高于Iose 386细胞,差异有统计学意义(P<0.05),并且Iose 386细胞中Trop-2蛋白表达水平不受影响。在HO8910细胞中,与PBS组相比,Trop-2组细胞存活率显著降低,且随着时间的增加,存活率逐渐降低,差异均有统计学意义(P<0.05)。与cisplatin组相比,Trop-2 IgG+cisplatin组中HO8910细胞和Iose 386细胞存活率都降低,差异有统计学意义(P<0.05)。在HO8910细胞中,与PBS组相比,其余3组细胞迁移和侵袭能力降低,并且Trop-2 IgG+cisplatin组中迁移和侵袭细胞数量少于cisplatin组,差异均有统计学意义(P<0.05);而在Iose 386细胞中无效果。结论抗Trop-2 IgG联合顺铂后可特异性识别卵巢癌细胞表面的Trop-2抗原,且能加强顺铂对HO8910细胞的抑制效果,为卵巢癌抗体-药物偶联物的开发和靶向治疗提供了思路。

Objective To investigate the effect of anti-Trop-2 IgG antibody combined with cisplatin on ovarian cancer epithelial cells.Methods Trop-2 plasmid was transfected into 293F cells and purified using Protein A affinity column to obtain human anti-Trop-2 IgG antibody.The transfection efficiency of Trop-2 plasmid was detected by enzyme linked immunosorbent assay(ELISA).The purification efficiency of anti-Trop-2 IgG antibody was identified by polyacrylamide(SDS-PAGE).The molecular interplay Biacore T100 was used to detect the anti-Trop-2 IgG antibody and Trop-2 antigen affinity with Trop-2 antigen.Protein immunoblotting(Western blotting),flow cytometry and immunofluorescence were used to detect the expression level of Trop-2 protein in HO8910 cells and Iose 386 cells.The experiments were divided into 4 groups,phosphate buffer solution(PBS)group,Trop-2 IgG group,cisplatin group and Trop-2 IgG+cisplatin group.CCK8(cell counting kit-8)was used to detect the survival rate of ovarian cancer cells,and Transwell was used to detect the migration and invasion ability of ovarian cancer cells.Results The human anti-Trop-2 IgG antibody was successfully transfected and purified,and it showed high binding and immunoreactivity to Trop-2.The anti-Trop-2 IgG antibody was highly expressed in HO8910 cells,but not in Iose 386 cells.When cisplatin was present,Trop-2 protein expression levels were higher in HO8910 cells than those in Iose 386 cells,the differences were statistically significant(P<0.05),and Trop-2 protein expression levels were unaffected in Iose 386 cells.In HO8910 cells,cell survival was significantly lower in the Trop-2 group compared with the PBS group,and the survival rate decreased gradually with increasing time,the differences were statistically significant(P<0.05).Both HO8910 cell and Iose 386 cell survival was reduced in the Trop-2 IgG+cisplatin group compared to the cisplatin group,the differences were statistically significant(P<0.05).In HO8910 cells,the migratory and invasive abilities of the remaining three groups of cells were reduced compared with the PBS group,and the number of migrating and invading cells was less in the Trop-2 IgG+cisplatin group than that in the cisplatin group,the differences were statistically significant(P<0.05),while there was no effect in Iose 386 cells.Conclusion The combination of anti-Trop-2 IgG with cisplatin specifically recognized Trop-2 antigen on the surface of ovarian cancer cells and enhanced the inhibitory effect of cisplatin on HO8910 cells,providing an idea for the development of antibody-drug couples for ovarian cancer and targeted therapy.