Adenovirus-mediated Gene Transfer Of Endostatin Resulting In Inhibition Of Lewis Lung Carcinoma Growth And Synergistic Effect With Chemotherapy

Anti-angiogenesis is a hot spot of tumor therapy currently. Endostatin, discovered by O'Reilly in 1997, has been closely studied due to its strong anti-angiogenic effect. Although endostatin has appeared to act effectively as an anti-tumor drug in animal models, its recombinated protein has shortcomings such as shorter therapeutic time, need for repeated administrating and its side effect. Thus, the present experiment constructed non-replicative adenovirus coding mouse endostatin(mES) gene. On the ground of observing it's transfection effect, reliability and anti-angiogenic activity in human umbilical vein endothelial cells (ECV304) in vitro, we observed it's inhibiting growth and metastasis of tumor and further studied the synergistic effect with cyclophosphamide on mouse lewis lung carcinoma models in vivo. No documents has been published on such a study till now. This provided vacuum for our expriment. So the present study proceed three parts:Partâ…  Construction of non-replicative adenovirus vector coding mouse endostatin gene (Ad-mES).Objective:To construct non-replicative adenovirus vector coding mouse endostatin gene (Ad-mES) and it's reliability was evaluated in vitro. Methods: Adenovirus acted as gene transferring vector. And mouse endostatin gene as therapy gene was cloned into shuttle plasmid by enzyme cutting and connecting and formed transfer plamid of pCA13-mES. Plamid of pCA13-mES were co-transfected into 293 cells with adenovirus genomic DNA plasmid of pBHGE3 by co-transfecting with catholyte and liposomes for package of replication-defective recombinant adenovirus particles(Ad-mES). PCR and enzyme cutting test indicated that the recombinant Ad contained the mES gene, and Ad-mES was amplificated in 293, purified by CsCl density gradient centrifugation. It's titer was assayed by TCID50. Meantime, it's reliability was evaluated by infecting Hela cell. Results:The target gene was inserted successfully into adenovirus vector pCA13. The replication-defective adenovirus coding endostatin gene was packaged by virus recombination technology. The titer of amplified and purified recombinant adenoviruses (Ad-mES) reached 1010pfu/ml. Hela cells grew well. Conclusion: We had constructed the recombinant adenovirus which was higher titer, better purity and lower toxicity.Partâ…¡ Transfecting (Lewis Lung Carcinoma)LLC cell by Ad-mES and inhibiting proliferation of endothelial cells by conditioned supernatant after infection in vitro.Objective: To observe adenovirus vector's transfection effect, reliability and anti-angiogenic activity in human umbilical vein endothelial cells (ECV304) in vitro. The experiment will settle groundwork for the further study in vivo. Methods: Utilizing adenovirus taking Green Fluorescence Protein (Ad-GFP) to transfect LLC for measuring infection efficiency and observing LLC'S growth and shape. LLC were infected by different multiplication of infection (MOI) by 100MOI at different duration(24h,48h,72h,96h). The expression of mouse endostatin was surveyed by immunohistochemical (IHC) staining and Western Blot after LLC were transfered 48 hours. ELISA assay was employed to estimate quantity of mouse endostatin in conditioned supernatant after transfering 24h,48h,72h and 96h. And the effect of inhibiting to ECV304 was assayed at different MOI by MTT(Methyl thiazolyl tetrazolium) . Results:80 percent of LLC can be infected effectively by Ad-GFP at 100MOI and the shape of these cells were normal. So the vector was testified to be effective and safe. After cell being infected 48 hours, endostatin was identified in the cell plasma of infected LLC by IHC and negative result were founded in non-infected LLC. We also observed the expression band of endostatin in 20 kDa in culture supernatant of infected LLC by Western Blot. Endostatin expressed in the supernatant in infected group and its concentration was climbing up with increasing MOI by ELISA assay. The expression of mES lasted for at least 96 hours. While negative results were found in the control group(p<0.01). MTT appear...