Aav Mediated Local Delivery Of Light Suppresses Tumorige-nesis In A Murine Cervical Cancer Model

AAV mediated local delivery of LIGHT suppresses tumor-igenesis in a murine cervical cancer model[Abstract] Objective To explore the feasibility of reverse effect of recombinant LIGHT adeno-associated virus(rAAV-LIGHT) in mouse cervical cancer model. Methods Using DNA recombination technique to construct AAV main plasmid LIGHT. rAAV-LIGHT and rAAV-GFP were produced by co-transfaction efficiency was measured.The titer was measured by Quantitative Real-time PCR .After transfactin rAAV-LIGHT to TC-1 cells the espression of LIGHT was measured by RT-PCR. Using lymphocyte cytotoxcity assay to test the immunity effects of LIGHT. Mouse cell line TC-1 (transfected by E6E7RAS) was used in the establishment of the cervical cancer model. Three groups of cervical cancer models applied by PBS(as control)(5 mice), rAAV-GFP(5 mice), rAAV-LIGHT(5 mice) intramuscularly on the 3d after innoculation.The biologic feature of the model was observed and the espression of LIGHT and other cytokine experession in the tumor microenvironment was measured by Real-time PCR. Then the effect of rAAV-LIGHT on supperssion tumor growth in models were observed. Analyzed the mice spleen lymphocyte proliferation by flow cytometry, Tumor tissue H&E and Immunohistochemistry. Results rAAV-LIGHT and rAAV-GFP were constructed and identified successfully.The titers were both 2.5×1010 virus particles/ml. The transfection efficiency was 60%. SPSS13.0 analysis showed rAAV-LIGHT group tumor size smaller than control and rAAV-GFP(P=0.0055,P=0.029 P < 0.05). The control and rAAV-GFP group showed no statistically difference in tumor size(P=0.115 P>0.05). Conclusion rAAV-LIGHT could reverse tumor growth in cenrvical cancer model. Construction and identification of recombinant mouse HVEM adeno-associated virus【Abstract】Objective To clone mouse HVEM gene into AAV vector pAAV-IRES-hrGFP and obtain high titer and purity recombinant mouse HVEM-AAV and infection of CHO cells for expression, to lay the foundation for immuno-gene therapy. Methods Framework plasmid of recombinant HVEM-AAV was constructed by gene recombination. The recombinant plasmid pAAV-IRES-HVEM-hrGFP , pAAV-RC ,pHelper were co-transfected into AAV-293 cells according to the calcium phosphate based protocol and produced recombinant virus rAAV-HVEM purified by PEG/chloroform. The purity of recombinant rAAV-HVEM was verified by SDS-PAGE. The titer of the virus particles was detected by Quantitative Real-Time PCR. HVEM expression in CHO cells which were infected by recombinant rAAV-HVEM was verified by RT-PCR and FACS analysis. Results Recombinant mouse rAAV-HVEM was successfully constructed with high titer and high purity. After transfection recombinant AAV-HVEM mediated a stable expression of HVEM mRNA in CHO cells。Conclusion Recombinant mouse rAAV-HVEM is successfully constructed, which will benefit for further study the function of HVEM and its applications.