Treatment Of Mouse Lung Carcinoma By Endostatin Gene Mediated By Adenovirus Vector

BackgroundAngiogenesis , the formation of new capillaries from the preexisting blood vessels, is critical for tumor growth and metastasis. A cancer cann't exceed the size of 2-3mm without angiogenesis. Inhibiting tumor angiogenesis is a promising new strategy for treating cancer. The target of antiangiogenic cancertreatment is the genetically normal endothelial cell. Therefore, the development of resistance to angiostatic therapy is very unlikely. Endostatin is a newly found potent endogenous inhibitor of angiogenesis. Endostatin can inhibit tumor growth completely and decrease tumor metastasis in animals. However, there are a number of potential limitations to endostatin in their recombinat forms. Endostatin is relatively unstable in vitro, require high doses for antitumor efficacy and need to be administered chronically. These limitations have prompted investigators to study gene therapy approaches to deliver endostatin . We chose the highly efficient adenovirus-based gene delivery system for our model to test whether adenovirus transfer of the endostatin gene could generate sufficient functional endostatin in vivo to inhibit tumor growth.Objective1. Construct recombinant adenoviral vector containing the whole murine endostatin gene.2. Observe the expression of Ad-mEndo and bioactivity in vitro, and delineated the mechanism of action.3. Observe the expression of Ad-mEndo in vivo, inhibition of tumor growth and metastasis, and the inhibition of tumor angiogenesis.Methods:I. We constructed recombinant adenoviral vector containing the full-length mouse endostatin cDNA.. Recombinant adenoviruses were generated bypackaging of HEK293 and purified by velocity density gradient centrifugation in caesium chloride solutions. The plaque-forming units (pfu) were determined by 50% tissue culture infection dose(TCID)2. Observe the infection rate of adenovirus in fluorescence microscopy, the expression of endostatin after transfection of LLC and ECV304 with Ad-mEndo at mRNA level by RT-PCR, the expression of endostatin protein in supernatant of viralhy transduced LLC and ECV304 cells by Western Blot, and endostatin conditioned supernatant level was determined by ELISA.3. A coiorimtric MTT was used to determine the proliferation of endothelial cell and non-endothelial cell transfected with Ad-mEndo. FACS was done to detect the change of cell cycle of LLC cell and ECV304 cell transfected with Ad-mEndo, and AnnexinV-FITC staining to quantify the percent of cell undergoing early apoptosis.4. Establish murine transplant model by subcutaneous inoculation 2 X106 LLC into the dorsa of C57BL/6 mice. Immunohistochemistry detecting the expression of endostatin in vivo after intratumor injection of Ad-mEndo. The level and continuation of expression were determined by ELISA and Western Blot.5. Observe the inhibition of tumor after injection of Ad-mEndo and the change of survival. Observe the MVD change staining by CD31 and CD105 antibody, and the tumor apoptosis under transmission eletron microscope.Results:1. We constructed the recombinant adenoviral vector containing the whole murine endostatin gene. The Ad-mEndo and Ad-GFP titer can reach 9.2 X 1011 and 7.8x1011respectively.2 Adenovirus can transfect LLC and ECV304 cell efficiently. The mRNA of mendostatin was detected in the infected LLC and ECV304 by RT-PCR. The mendostatin protein was 3275 ?505ng/ml in the infected LLC cell culture supernatant. Ad-mEndo infecting can inhibit the proliferation of ECV304 ,There was no growth inhibition on LLC infected by Ad-mEndo at MOI 100. Endostatin can block the stimulus of VEGF165 to endothelial cell, and cause G1 arrest and apoptosis of endothelial cells.3. In contrast with control group, intratumor injection of Ad-mEndo can inhibit the growth and metastasis of tumor significantly, and prolong the survival rate of mice. Strong expression of mendostatin was seen in the tumor tissue after injection of Ad-mEndo immunostained by mouse endostatin monoclonal antibody, wh...