CAJ | 학술논문

目的：探讨抗明胶酶单链抗体 Fv 与力达霉素（LDM）融合蛋白的抗肿瘤作用机制及疗效。方法采用三联径向加压 C4柱制备 LDM 发色团 AE，体外分子重建将其装入融合蛋白 Fv-LDP 中，Superdex 75层析获得 Fv-LDP-AE。反相 HPLC 测定纯度或相对含量。流式细胞术测定 DNA 含量；采用 SA-β-gal 染色、Western blot 和细胞伤口愈合实验分别检测药物对细胞衰老、蛋白表达和迁移的影响；通过动物模型评价药物疗效。结果 Fv-LDP-AE 承袭了 LDM 诱导肿瘤细胞周期阻滞、凋亡和裂亡的作用特点，并显示出更强的诱导衰老和抗迁移作用。Fv-LDP-AE 诱导凋亡和抑制迁移依次与 caspases 通路激活和 MMP-2降调节有关。Fv-LDP-AE 可抑制小鼠和裸鼠移植瘤生长，抑制率分别可达到86.6%和82.5%。结论 Fv-LDP-AE 对小鼠和裸鼠移植瘤有效，可能与其对细胞周期阻滞、凋亡、裂亡和衰老的诱导以及迁移的抑制有关。
목적：탐토항명효매단련항체 Fv 여력체매소（LDM）융합단백적항종류작용궤제급료효。방법채용삼련경향가압 C4주제비 LDM 발색단 AE，체외분자중건장기장입융합단백 Fv-LDP 중，Superdex 75층석획득 Fv-LDP-AE。반상 HPLC 측정순도혹상대함량。류식세포술측정 DNA 함량；채용 SA-β-gal 염색、Western blot 화세포상구유합실험분별검측약물대세포쇠로、단백표체화천이적영향；통과동물모형평개약물료효。결과 Fv-LDP-AE 승습료 LDM 유도종류세포주기조체、조망화렬망적작용특점，병현시출경강적유도쇠로화항천이작용。Fv-LDP-AE 유도조망화억제천이의차여 caspases 통로격활화 MMP-2강조절유관。Fv-LDP-AE 가억제소서화라서이식류생장，억제솔분별가체도86.6%화82.5%。결론 Fv-LDP-AE 대소서화라서이식류유효，가능여기대세포주기조체、조망、렬망화쇠로적유도이급천이적억제유관。
Objective To investigate the antitumor mechanism and efficacy of the fusion protein Fv-LDP-AE composed of lidamycin (LDM) and anti-gelatinases single-chain Fv antibody. Methods Free AE (the active enediyne chromophore of LDM) prepared using the triple radial pressure C4 column was reassembled into fusion protein Fv-LDP by in vitro molecular reconstitution. The rebuilt fusion protein Fv-LDP-AE was obtained by Superdex 75 chromatography to remove the free AE. Reverse phase HPLC was used to determine the purity or the relative content of the drug components. DNA content was measured by flow cytometry. SA-β-gal staining, Western blot and cell wound healing assay were exploited to detect the effect of tested drugs on cell senescence, protein expression and cell migration, respectively. Antitumor efficacies of tested drugs were evaluated by different animal models. <br> Results Fv-LDP-AE inherited the action characteristics derived from LDM including the induction of cell cycle arrest, apoptosis and mitotic cell death in human hepatoma BEL-7402 and lung cancer PG-BE1 cells. Furthermore, Fv-LDP-AE also showed more potent activity than LDM in triggering cellular senescence and inhibiting cell migration in human cancer cells. The induction of cell apoptosis was related to the activation of caspases signaling pathway and the inhibition of migration was associated with the down-regulation of MMP-2 in Fv-LDP-AE-treated BEL-7402 cells. In vivo, Fv-LDP-AE also suppressed the growth of murine tansplantable hepatoma 22 (H22) and human hepatoma BEL-7402 xenograft in athymic mice with the inhibition rate of 86.6%and 82.5%, respectively. Conclusion Fv-LDP-AE showes antitumor effects against murine transplantable tumor and cancer xenograft in athymic mice, which may be related to the induction of cell cycle arrest, death and senescence as well as the inhibition of cell migration by Fv-LDP-AE in tumor cells.