Abstract:

Objective To observe the bioactivity of adenovirus-mediated human endostatin gene in vivo and in vitro. Methods B16F10 melanoma cells and human endothelial cells （ECV 304） were both transfected with recombinant adenovirus containing green fluorescent protein （Ad-GFP） or human endostatin gene （Ad-mES） at various multiplicity of infection （MOI）. Then, the expression of endostatin gene was detected by RT-PCR, and the growth of cells by MTT assay. B16F10 cells were inoculated into the back of mice to establish melanoma models, which were classified into treated groups intratumorally injected with Ad-mES once （single Ad-mES group） or repeatedly （repetitive Ad-mES group） with an interval of 7 days, and control groups intratumorally injected with Ad-GFP （Ad-GFP group） or phosphate buffered solution （PBS group）. Subsequently, the growth of tumors was observed at an interval of 4 days; tumor tissue samples were obtained from killed mice and subjected to the detection of endostatin expression with immunohistochemistry on day 7 after the first intratumoral injection; transmission electron microscopy was used to observe the apoptosis of tumor cells and endothelial cells on day 14. Results Adenovirus could efficiently infect targeted cells. When the MOI of Ad-GFP was 10, 20, 50, 100, 200 and 500, B16F10 cells were infected at a rate of 15.6%, 35%, 73%, 88%, 95.2% and 97%, respectively, and ECV304 cells at a rate of 19%, 35%, 80%, 90%, 97% and 98.5%, respectively. The endostatin gene was proved to be expressed in targeted cells. Ad-mES had no obvious effect on B16F10 cells, but inhibited the growth of ECV 304 cells, and the inhibitive effect was enhanced with the concentration of Ad-mES. The rate of tumor formation was 100% on day 8 after injection of B16F10 cells, and the earliest death of mice was observed on day 16 in PBS group, day 18 in Ad-GFP group, day 20 in single Ad-mES group and repetive Ad-mES group. Conclusions The recombinant Ad-mES could affect the bioactivity of targeted cells in vivo and in vitro. Intratumoral injection of Ad-mES prolonged the survival of mice bearing melanoma and deccelarated the growth of melanoma.