Research On CD44v6 And VEGF Expression In ACC-M Transfected With AD-GFP-nm23-H1 & AD-GFP-nm23-H1 Toxicity Test In Vivo

Objective:Based on previous studies, took the cell line ACC-M cultured in vitro and a recombinant adenoviral containing nm23-hl (Ad-GFP-nm23-H1) as research objects, and observed the regulation of cancer related genes CD44V6 and VEGF after Ad-GFP-nm23-H1 transfected to ACC-M cell; At the same time made animal toxicity test to evaluate its safety. We hope this study can provide some theory and method basis for more advanced primate studies and gene therapy studies.Methods:1.Growing cells were treated with Ad-GFP-nm23-H1 at a final concentration of 10⁸PFU/ml,10⁹PFU/ml,10¹⁰PFU/ml respectively. Observe the transfection after being treated for 12,24h under the fluorescence microscope.2.PCR detection of NM23-H1 expressed by Ad-GFP-nm23-H1 in ACC-M cell line, and semiquantitative analysis of VEGF and CD44v6 mRNA levels by RT-PCR.3.Assay expression levels of VEGF and CD44v6 by immunohistochemistry.4.Experiment in vivo,100μl,200μl and 400μl recombination adenovirus and 200μl PBS were injected into kunming mouse for nine times in three weeks. Test liver and kidney function of each group in every week. At termination, kill all mouse of each group, observe virus distribution and nm23-hl expression in vivo by frozen section, HE pathological section and immunohistochemi-stry.Result:1.Ad-GFP-nm23-Hl of 10⁹PFU/ml and 10¹⁰PFU/ml had strong transfection ability on ACC-M cells.2.The expression level of nm23-H1 in ACC-M treated with Ad-GFP-nm23-Hl is higher than treated with PBS and empty virus.3.The mRNA level of VEGF and CD44v6 in ACC-M treated with Ad-GFP-nm23-H1 i s lower than treated with PBS and empty virus. 4.The expression level of VEG F and CD44v6 in ACC-M treated with Ad-GFP-nm23-Hl is lower than treated with PBS and empty virus.5.Occasionally, liver and kidney function is mildly ab normal in Ad-GFP-nm23-Hl groups. But there is no significant correlation betwe en incidence rate and dose levels.6.The major change are hadropic degeneration, focal necrosis and infiltration of inflammatory cells around central vein.7. The frozen sections show that adenoviruse were mainly distributed in liver and lung tissues.8.Immunohistochemistry results show that nm23-H1 expression level was high in liver and lung.9.The antibody levels reached its peak in third week. T he serum neutralization antibodies could block 5×10⁶ pfu adenovirus infection. After stop injection four weeks, antibody level began to decrease.Conclusion:1.ACC-M cell can be transfected with high transfection rate by Ad-GFP-nm23-H1;2.Nm23-H1 can express effiiciently and stablely in the ACC-M cell;3.VEGF,CD44V6 can be significantly downregulated after Ad-GFP-nm23-hl transfection ACC-M cell;4. Mice can tolerant the Ad-GFP-nm23-H1, Occasional liver function abnormalities and liver damage and other minor side effects, no other significant side effect;5.Repeat injection Ad-GFP-nm23-Hl can induce a high titer of anti-Adenovirus antibodies,But can fall within a short time after stopping injection.