【摘要】 目的研究靶向EGFR/HER-2的融合蛋白Ec-LDP-Hr及其烯二炔强化融合蛋白Ec-LDP-Hr-AE对结肠癌的抗肿瘤活性。方法 ELISA和细胞免疫荧光法鉴定融合蛋白与结肠癌HCT-15的结合活性。克隆形成实验和MTT法检测Ec-LDP-Hr对结肠癌HCT-15细胞的增殖活性影响,并用Western blot法检测EGFR/HER-2的表达量改变。用MTT法和流式细胞术检测强化融合蛋白Ec-LDP-Hr-AE对HCT-15的细胞毒性及诱导凋亡情况。体内实验研究融合蛋白Ec-LDP-Hr、强化形式Ec-LDP-Hr-AE及其联合对裸鼠结肠癌移植瘤的抑瘤效果。结果结肠癌HCT-15、HCT-116和HT-29细胞都显示较高的EGFR和HER-2表达量。融合蛋白Ec-LDP-Hr能与结肠癌HCT-15细胞结合,并可通过下调靶点EGFR/HER-2的表达从而抑制HCT-15细胞的克隆形成和细胞增殖。强化融合蛋白Ec-LDP-Hr-AE对HCT-15有很强的细胞毒作用,并且在低浓度下即可诱导细胞的凋亡。动物实验结果显示融合蛋白和强化融合蛋白对结肠癌HCT-15裸鼠移植瘤的抑瘤率分别为和37.6%和56.7%,两者联合可提高抑瘤率至71.4%。结论靶向EGFR/HER-2的双特异性融合蛋白Ec-LDPHr对结肠癌HCT-15的裸鼠移植瘤有一定的抑瘤作用,联合强化融合蛋白Ec-LDP-Hr-AE能提高抗结肠癌疗效。更多还原

【Abstract】 Objective To evaluate the antitumor efficacy of the EGFR /HER- 2 bi- targeting fusion protein Ec- LDP- Hr and its enediyne- energized fusion protein Ec- LDP- Hr- AE against colorectal cancer. Methods Fusion protein Ec- LDP- Hr was expressed in E. coli and purified by affinity chromatography. The expressional levels of EGFR and HER- 2 in various colorectal cell lines were detected by Western blot. ELSIA and cell immunofluorescence assay were used to investigate the binding activity of fusion protein Ec- LDP- Hr to EGFR /HER- 2 overexpressed colorectal cancer cells. MTT and colony formation assay were used to investigate the anti-proliferation effect of the fusion protein on cancer cells. The cytotoxicity and apoptosis induced by enediyne- energized Ec- LDP- Hr-AE were analyzed by MTT and flow cytometry respectively. Therapeutic efficacy was evaluated with colorectal cancer HCT- 15 xenograft in athymic mice. Results High expressional levels of EGFR and HER- 2 were detected in all tested colorectal cancer cell lines,including HCT- 15,HCT- 116 and HT- 29. The targeting fusion protein Ec- LDP- Hr had strong binding activity to the EGFR /HER- 2 overexpressing HCT- 15 cells. Fusion protein Ec- LDP- Hr also inhibited the proliferation of HCT- 15 cells by down- regulating the expression of EGFR /HER- 2. The energized fusion protein Ec- LDP- Hr- AE showed highly potent cytotoxicity to HCT- 15 cells. Moreover,it could induce the cell apoptosis at very low concentrations. In vivo,Ec- LDP- Hr or Ec- LDP- Hr- AE alone suppressed tumor growth by 37. 6% and 56. 7%,respectively. The combination of them suppressed tumor growth up to 71. 4%. Conclusion EGFR /HER- 2 targeting protein Ec- LDP- Hr had strong binding ability and proliferation- inhibiting activity to cancer cells. The combination of fusion protein Ec- LDP- Hr and enediyne- energized fusion protein Ec- LDP- Hr- AE might be a new strategy for enhancing antitumor efficacy against colorectal cancer.更多还原