Ribozyme mediated cleavage of cell-associated isoform of vascular endothelial growth factor (VEGF) inhibits liver metastasis of a pancreatic cancer cellline

间质血管生成是恶性肿瘤进展的重要因素。我们使用锤头状核酶对抗血管内皮生长因子(VEGF)基因转录，下调胰腺癌细胞系MIA-PaCa2的细胞相关VEGF189亚型功能。通过脂质体转染法将b-肌动蛋白启动子驱动的核酶序列导入细胞。我们评估了它们对细胞系生长和新生血管的影响。通过将转化子注射到裸鼠的脾髓内来评估转移潜能。体外实验表明，抗VEGF189核酶能特异性有效地切割VEGF189的mRNA异构体，但不能切割其他类型的异构体。在组织培养条件下，引入抗VEGF189核酶的MIA-Paca2细胞的生长速率与引入去能核酶的MIA-Paca2细胞相比没有发生任何变化。人脐血管内皮细胞与导入抗VEGF189的MIA-Paca2细胞共培养时，组织型纤溶酶原激活剂的产生减少。当转化子被皮下移植时，核酶导入的MIA-Paca2异种移植瘤的肿瘤体积明显小于禁用的核酶导入的异种细胞系(P&lt；0.01，Dunnett‘s检验)。反义VEGF189的MIA-Paca2组无明显转移，而去功能化核酶MIA-Paca2组有明显的肝转移(P&lt；0.05，Fisher‘s精确检验)。这些结果提示，VEGF189通过改变肿瘤组织中的血管生成平衡，在肿瘤的生长和转移潜能中发挥重要作用。

Stromal angiogenesis is an important factor for progression of malignant neoplasms. we used hammerhead ribozymes against vascular endothelial growth factor (VEGF) gene transcripts to downregulate cell-associated VEGF189 isoform function in a pancreatic cancer cell line MIA-PaCa2. The b-actin promoter-driven ribozyme sequences were introduced into the cells by lipofection. We evaluated their effects on growth and neovascularisation of the cell lines. Metastatic potential was evaluated by the injection of transformants into the splenic pulp of nude mice. Anti-VEGF189 ribozyme specifically and efficiently cleaved the VEGF189 mRNA isoform but not other types of isoforms in vitro. MIA-Paca2 introduced with anti-VEGF189 ribozyme did not show any alteration of growth rate under tissue culture conditions compared to MIA-Paca2 introduced with disabled ribozyme. Production of tissue type plasminogen activator decreased in the human umblical vascular endothelial cells cocultured with the MIA-Paca2 cell line introduced with anti-VEGF189. When the transformants were subcutaneously transplanted, tumour volume of the ribozyme-introduced MIA-Paca2 xenografts was significantly smaller than that of disabled ribozyme-introduced cell line xenografts (P<0.01, Dunnett's test). No metastasis was apparent of the MIA-Paca2 introduced with anti-VEGF189, while disabled ribozyme transfected MIA-Paca2 show significant liver metastasis (P<0.05, Fisher's exact test). These results suggested that the VEGF189 mRNA plays an important role in growth and metastatic potential through alteration of angiogenic balance in cancer tissue.