二硫键稳定的人源性抗bFGF双链抗体抑制人肺癌细胞生长的作用研究

The effect of a disulfide-stabilized diabody against bFGF on the growth of human lung cancer

目的研究二硫键稳定的人源性抗b FGF双链抗体(ds-Diabody)对人肺癌A549细胞的体内外增殖抑制作用。方法利用镍离子亲和层析和离子交换层析的方法纯化得到ds-Diabody,间接ELISA检测其抗原结合活性,CCK-8法检测ds-Diabody对人肺癌A549细胞的增殖抑制作用,Western blot分析其作用于肿瘤细胞的机制,Transwell检测其对肿瘤细胞迁移侵袭的影响。建立裸鼠皮下移植瘤模型,记录各处理组裸鼠皮下移植瘤的体积及裸鼠体质量变化。免疫组化法检测肿瘤组织CD31和LYVE1的表达情况,初步探讨抗b FGF的人源性ds-Diabody抗体的抗肿瘤机制。结果成功纯化得到具有抗原结合活性的ds-Diabody。CCK-8结果显示,纯化的ds-Diabody可剂量依赖性地抑制人肺癌A549细胞株的增殖。Western blot结果表明ds-Diabody能有效阻断与b FGF相关的MAPK和Akt通路。Transwell结果表明ds-Diabody可以显著抑制A549细胞的迁移及侵袭。裸鼠体内试验中,ds-Diabody抗体显著地抑制了肿瘤的生长,抑制率为86.54%。免疫组化的结果显示ds-Diabody抗体处理组肿瘤组织中微血管密度和淋巴管密度均有下降。结论抗bFGF的人源性ds-Diabody抗体可有效地抑制人肺癌A549细胞的生长。

This study designed to investigate the effects of the disulfide-stabilized diabody against bFGF （ds-Diabody） on the growth of human lung cancer A549 ceils in vitro and in vivo. Expressed ds-Diabody was purified by affinity chromatography of Ni-Seproase 6 FF and ion exchange chromatography of DEAE Sepharose FF. The results of ELISA showed that the purified ds-Diabody could specifically bind to bFGF; CCK-8 showed that the purified ds-Diabody could inhibit the proliferation of A549 cells in a dose-dependent manner in vitro. Western blot showed that the ds-Diabody could effectively block bFGF/FGFR-triggered the signal pathway of Akt and MAPK. The results of transwell showed that the ds-Diabody could significantly inhibit the migration and invasion of A549 cells. The results of nude mice experiments showed that the ds-Diabody could significantly inhibit the growth of lung cancer in mice and inhibition rate was 89.37%. The immunohistochemistry showed that the microvessels and lymphatic vessels were significantly reduced in the group of the ds-Diabody. All the results demonstrated that the Disulfide-stabilized diabody against bFGF could effectively inhibit the growth of human lung cancer A549 cells.