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目的:筛选、鉴定抗人肝癌血管内皮功能性单抗,为治疗肝癌提供靶向治疗剂,并为分离获得肝癌相关的分子靶标打下基础。方法:采用活细胞荧光、MTT细胞增殖实验、成管实验和动物体内治疗实验,筛选鉴定抑制肝癌内皮细胞的功能性单抗。结果:从能与肝癌内皮细胞膜反应的119株克隆中,筛选出16株单抗显著地抑制肝癌内皮增殖其抑制率达21%~46%,46株能显著抑制肝癌内皮细胞的成管,3株单抗1F9、12B5和1B11能显著抑制肝癌移植瘤的生长抑制率分别为50.8%、48.7%和47.0%。选择1株体内抑瘤效果最好的1B11单抗,通过抗体抗原亲和层析法进行纯化,并对纯化的单抗的相关抗原蛋白进行免疫组织化学实验鉴定,结果显示1B11抗原在肝癌血管组织较高的表达而在正常肝血管组织极少表达。蛋白质印迹法显示其抗原相对分子质量约46×103。结论:采用大容量功能性抗体库技术成功获得了多株具有抑制肝癌内皮恶性生物学行为的功能性单抗,体内外抑制肺癌的生长,具有成为肺癌靶向治疗剂的潜力。其中1株可能是一个靶向治疗肝癌的新靶位。
OBJECTIVE: To screen and identify functional monoclonal antibodies against human hepatocellular carcinoma vascular endothelial cells, to provide targeted therapeutic agents for the treatment of liver cancer, and to lay the foundation for the isolation of molecular targets related to liver cancer. Methods: Live mAb, MTT cell proliferation assay, tube-forming assay and in vivo animal experiment were used to identify and identify functional monoclonal antibodies that inhibit HCC endothelial cells. RESULTS: Of the 119 clones that reacted with HCC endothelial cell membrane, 16 monoclonal antibodies were screened to significantly inhibit the proliferation of HCC. The inhibition rate was 21% -46%. 46 of them could significantly inhibit the formation of HCC endothelial cells.3 Strains MAb 1F9, 12B5 and 1B11 could significantly inhibit the growth inhibition rates of hepatocellular carcinoma transplanted tumors to 50.8%, 48.7% and 47.0%, respectively. One of the 1B11 monoclonal antibodies with the best antitumor activity in vivo was selected and purified by antibody antigen affinity chromatography. Immunohistochemistry was performed on the relevant antigenic protein of the purified monoclonal antibody. The results showed that the 1B11 antigen was expressed in hepatocellular carcinoma tissue Higher expression is rarely expressed in normal hepatic vascular tissue. Western blot showed that the relative molecular mass of antigen was about 46 × 103. CONCLUSION: A large number of functional monoclonal antibodies that inhibit the malignant behavior of human hepatocellular carcinoma have been successfully obtained using large-scale functional antibody library. They can inhibit the growth of lung cancer in vitro and in vivo and have the potential of being targeted therapy for lung cancer. One of them may be a new target for the treatment of liver cancer.