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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 anti-human growth inhibition of human fibrosarcoma HT-1080 by anti-gelatinase dFv-LDP and its enhanced fusion protein dFv-LDP-AE. Methods The expression of gelatinase in different cell lines was analyzed by Western blotting. The growth inhibition of HT-1080 by fusion protein dFv-LDP and the enhanced fusion protein dFv-LDP-AE was analyzed by MTT method. Immunofluorescence and immunofluorescence analysis of fusion protein dFv-LDP and HT-1080 binding capacity; flow cytometry analysis of the two alone or in combination on the HT-1080 cycle arrest; evaluation of the fusion protein dFv-LDP and dFv-LDP- AE alone or in combination with anti-HT-1080Luc lung metastases. Results The abundance of gelatinase in HT-1080 was significantly higher than that of other tumor types. Enzyme-linked immunosorbent assay and immunofluorescence analysis showed that the fusion protein dFv-LDP had a good affinity with HT-1080 cells. Tetramethylbenzothiazolyl tetrazolium The results showed that dFv-LDP-AE had a strong killing effect on the proliferation of HT-1080 in vitro; FACS showed that the combination of dFv-LDP and dFv-LDP-AE had no synergistic effect on cell cycle arrest; The results of lung metastasis showed that the number of pulmonary metastases in the group of fusion protein dFv-LDP (10 mg.kg-1) was 55.8% (P <0.01) in the control group, while that of the enhanced fusion protein dFv-LDP-AE was between 0.4 and 0.6 mg The number of metastatic lesions at the dose of 1 kg kg-1 was 41.4% in the control group (P <0.05 vs. dFv-LDP 10 mg.kg-1) and 25.1% (in comparison with dFv-LDP 10 mg.kg-1 (P <0.05 compared with dFv-LDP-AE 0.4 mg.kg-1 group) and 13.1% (P <0.05). The number of lung metastases in combination with dFv-LDP was 20.3% in the control group (P <0.05 compared to dFv-LDP-AE 0.6 mg.kg-1 group), suggesting that combined administration may further enhance the anti-metastatic effect. Conclusions The fusion protein dFv-LDP combined with the enhanced fusion protein dFv-LDP-AE has a synergistic effect on the suppressive effect of experimental lung metastasis in HT-1080 in nude mice.