人纤维蛋白溶酶原kringle5(K5)抑制小鼠肝癌的血管生成和肿瘤生长

Inhibition of K5 on Neovascularization and Growth of Mouse Hepatocarcinoma

【目的】体内、体外实验观察人纤维蛋白溶酶原kringle5(K5)对肝癌血管生成和肿瘤生长的影响。【方法】大肠杆菌中表达,组氨酸结合柱亲和层析、纯化获得K5蛋白,SDS-PAGE和Westernblot方法鉴定K5蛋白的表达;MTT法分析K5对牛视网膜毛细血管内皮细胞(BRCEC)和肝癌细胞株Bel-7402、HepG2增殖的影响;建立皮下种植肝癌鼠模型,瘤内注射K5,检测抑瘤率及肝癌组织微血管密度(MVD)。【结果】SDS-PAGE及Westernblot鉴定获得K5纯化蛋白;K5特异性地抑制BRCEC的增殖,IC50=160nmol/L,而对肝癌细胞株Bel-7402、HepG2的增殖无抑制作用;K5显著抑制小鼠肝癌生长,治疗组肝癌组织MVD明显低于对照组。【结论】K5抑制移植性小鼠肝癌血管生成和肿瘤生长,特异性抑制血管内皮细胞的增殖可能是K5抑制肝癌血管生成的主要机制。K5具有治疗肝癌的潜在临床价值。

[Objective ]To investigate the effects of human plasminogen kringle 5(K5) on the neovascularization and growth of hepatocarcinoma, in vitro and in vivo.[Methods]K5 was expressed in E. coli BL-21(DE3) induced by IPTG and purified by Ni2+-His Bind Resin affinity chromatography. The purity and identity of K5 is examined by SDS-PAGE and Western blot analysis. The anti-proliferative effects of K5 on primary bovine retinal capillary endothelial cell (BRCEC),hepatoma Bel-7402 and HepG2 cell lines were examined by MTT assay. To evaluate K5 activity in vivo, the mouse hepatocarcinoma xenograft model was established by injection of mouse hepatoma cells subcutaneously into the oxter of Kunming mice. The mice then received a direct intratumoral injection of K5 or PBS as control. The tumor suppressing rate and microvessel density (MVD) in hepatoma tissues were assayed.[Results]The purity of recombinant K5 was over 90% according to the analysis of SDS-PAGE and Western blot analysis. K5 inhibited proliferation of primary endothelial cells in dose-dependent manner, IC50 is 160 nmol/L, but no effect on Bel-7402 and HepG2 tumor cell lines, suggesting cell type-specific inhibition. K5 significantly inhibited the growth of hepatocarcinoma xenograft and decreased microvessel density (MVD) in hepatoma tissues, compared with PBS control group(P< 0.05). [Conclusion]K5 inhibits the neovascularization and the growth of hepatocarcinoma in xenografted hepatocarcinoma model of Kunming mice. The effect may be associated with its specifically anti-proliferation on endothelial cell. These results suggest that angiogenesis inhibitor of K5 could have therapeutic benefits in hepatocarcinoma.