Adenovirus Mediated Transfer Of Angiostatin K1-5 Gene Inhibits Nude Murine Ovarian Carcinoma

It is well known that ovarian cancer and metastatses growth are angiogenesis-dependent. VEGF and orther factors can promote angiogenesis and tumor metastasis. At present, the studies on gene therapy are the dificulty point and focus point about the treatment of ovarian cancer. We are going to explore how to link the gene therapy and anti-angiogenesis gene therapy in order to find a new therapteutic methord and conception - anti-angiogenesis gene therapy - for treatment of ovarian cancer. Angiostati k1-5 is one of the strongest anti-angiogenesis factors up to now. The study's aims are to investigate the function of suppression to proliferation and migration for vascular endothelial cells and evaluate therapeutic potential of angiostatin kl-5 for nude murine ovarian carcinoma on the bases of constructing adenovirus vector with agiostatin k14-5 gene.Part 1The study on construction of adenovirus vector with the gene angiostatin k 1-5Objective To construct adenovirus vector with angiostatin kl-5 gene for investigating the function of suppression to proliferation and migration for human vascular endothelial cells. Methods With the use of gene recombination and clone technology, we construct the adenovirus vector with the gene angiostatin kl-5. Ad-angiostatin kl-5 was isolated from a single plaque, expanded in 293 cells and purified by cesium chloride centrifugation. Results Tissue culture infectious dose 50(TCID₅₀) indicated that the condence of resultant viruses was 1.5 × 10⁹ PFU /ml. It was purified by CsCL banding,final yielde were generally 1.1 × 10¹0 PFU /ml. Conclusions We successfully constructed adenoviruse vector with the gene angiostatin k1-5 and believed this work would be the base of the functional experiments.Part 2The construction of adenovirus vector with agiostatinkl-5 gene and the study of the function of suppression to proliferation and migration for human vascular endothelial cellsObjective To investigate the function of suppression to proliferation and migration for vascular endothelial cells (ECV-304). Methods On the base of suscessfully constructing adenovirus vector with agiostatin kl-5 gene, we are going to explore the function of suppression to proliferation and migration for vascular endothelial cells. In vitro vascular endothelial eclls proliferation assay and migration activity were investigated through direct infection, MTT and transwell chemotaxis assay. Each experiment was repeated three times and mean values were counted. Results 50%TCID indicated that the condence of resultant viruses was 1.5 X 109pfu/mll. It was purified by CsCL banding,final yield were generally 1.1 X 1010 pfu/ml. Through indirect infect assay and MTT, we found angiostatin kl-5 could inhibit human vascular endothelial cells proliferation. We utilized human vascular endothelial cells to study the effect angiostatin kl-5 on cell migration, the result showed that adenoviruse vector with angiostatin kl-5 could significantly inhibited ECV-304 migration. Conclusions We successfully constructed adenoviruse vector with angiostatin kl-5 and showed in vitro it could inhibit proliferation and migration of HUVEC.Part 3Adenovirus mediated transfer of angiostatin kl-5 gene inhibits nude murine ovarian carcinomaObjective To evaluate therapeutic potential of angiostatin kl-5 for nude murine ovarian carcinoma. Methods After construction of adenovirus vector expressing angiostatin kl-5 gene, we examine the inhibition of angiostatin kl-5 for HUVEC in vitro. On the other hand,adenovirus expressing angiostatin kl-5...