抗明胶酶单链抗体和力达霉素融合蛋白抗纤维肉瘤HT-1080增殖和转移的实验研究

Inhibitory Effects of Anti-Gelatinases scFv and Lidamycin Fusion Protein on Proliferation and Metastasis of Human Fibrosarcoma HT-1080

目的观察抗明胶酶dFv-LDP以及其强化融合蛋白dFv-LDP-AE对人纤维肉瘤HT-1080的生长抑制作用。方法采用蛋白质印迹法分析不同细胞系中明胶酶表达情况,四甲基偶氮唑蓝法分析融合蛋白dFv-LDP以及强化融合蛋白dFv-LDP-AE的对HT-1080的生长抑制作用;酶联免疫吸附法以及免疫荧光分析融合蛋白dFv-LDP和HT-1080的结合能力;流式细胞术分析两者单独或者联合对HT-1080的周期阻滞;评价融合蛋白dFv-LDP和dFv-LDP-AE单独和联合体内抗HT-1080Luc肺转移的活性。结果 HT-1080的明胶酶表达丰度较之其他类型肿瘤明显,酶联免疫吸附法和免疫荧光分析表明融合蛋白dFv-LDP与HT-1080细胞有着良好的亲和力;四甲基偶氮唑蓝法结果显示dFv-LDP-AE对HT-1080体外增殖有着强烈的杀伤作用;FACS表明融合蛋白dFv-LDP和dFv-LDP-AE的联合对细胞周期的阻滞没有明显的增效作用;体内实验性肺转移实验结果表明,融合蛋白dFv-LDP(10 mg.kg-1)组肺部转移灶数目为对照组的55.8%(P<0.01);强化融合蛋白dFv-LDP-AE在0.4和0.6 mg.kg-1剂量下的转移灶数目分别为对照组的41.4%(与dFv-LDP 10 mg.kg-1组相比,P<0.05)和25.1%(与dFv-LDP10 mg.kg-1组相比,P<0.05);联合dFv-LDP后,其肺部转移灶数目为对照组的20.3%(与dFv-LDP-AE 0.4 mg.kg-1组相比,P<0.05)和13.1%(与dFv-LDP-AE 0.6 mg.kg-1组相比,P<0.05),提示联合给药可进一步提高抗转移的效果。结论融合蛋白dFv-LDP联合强化融合蛋白dFv-LDP-AE对裸鼠移植HT-1080实验性肺转移的抑制效果具有增效作用。

OBJECTIVE To investigate the antitumor efficacy of anti-gelatinases dFv-LDP and its enediyne-energized fusion protein dFv-LDP-AE on human fibrosarcoma HT-1080 cancer cells. METHODS Western blot was used to analyze the expression level of gelatinases in different cancer cell lines. The inhibitory effects of fusion protein dFv-LDP and its enediyne-energized fusion protein dFv- LDP-AE on HT-1080 were determined by MTF assay. The binding capability of fusion protein dFv-LDP with HT-1080 was detected by ELISA and immunofluorescence. FACS was used to analyze the cell cycle arrest by dFv-LDP, dFv-LDP-AE or their combination on HT- 1080 cells. The anti-metastasis effects of dFv-LDP, dFv-LDP-AE or their combination on the experimental lung metastasis model estab-lished by HT-1080Luc via tail vein injection were also evaluated in this study. RESULTS Expression level of gelatinases was higher in HT-1080 cells as compared to that of other cancer cell lines. The fusion protein dFv-LDP showed well binding capability with HT-1080 cells as determined by ELISA and immunofluorescence. The enediyne-energized fusion protein dFv-LDP-AE displayed extremely inhibitory effect on proliferation of HT-1080. Results of FACS indicated that the combination of dFv-LDP with dFv-LDP-AE could not further increase the GJM proportion on cell cycle arrest. However, in vivo experiment as examined using the experimental lung metastasis model established via HT-1080Luc tail veil injection, the metastasis foci in group of fusion protein dFv-LDP （ 10 mg · kg-1 ） was 55.8% compared to that of control group （ P 〈0.01 ）. The metastasis foci in group of dFv-LDP-AE at dosage of 0. 4 and 0. 6 mg · kg-1 were 41.4% and 25.1% respectively compared to that of dFv-LDP 10 mg · kg-1 group （P 〈0. 01 ）. The combination of dFv-LDP （ 10 mg · kg-1 ） with dFv-LDP-AE （0. 4 or 0. 6 mg· kg-1 ） showed an additive decrease of metastasis foci number in the lung of athymic mice, which were 20. 3% （P 〈0. 05 ,compared with dFv-LDP-AE at 0. 4 mg · kg-1 ） and 13.1% （P 〈0. 05 ,compared with dFv-LDP-AE at 0. 6 mg· kg-1 ） respectively. CONCLUSION The combination of dFv-LDP with its enediyne-energized fusion protein dFv-LDP-AE would inten