Construction Of Recombinant IL-7/CCL21 Adenovirus And Its In Vivo Study On Mouse Melanoma

PART1 Construction of pAdenoG-cmv-IL-7-sv40-CCL21 adenovirus vectorObjective To construct a recombinant adenovirus vector co-express IL-7 and CCL21in mouse melanoma B16F10 cells,providing a basis for subsequent experiments.Methods PCR technology was used to obtain the aim gene fragment and to construct IL-7-CCL21-pDONR plasmid vector.Then competent cells transformed the cells with IL-7-CCL21-pDONR plasmid vector.HEK293 cells were transfected with the vector,positive clones and sequencing analysis.Adenovirus vectors packaged and the titer was determined.Then the recombinant adenovirus was used to infect B16F10cell.Real-time PCR and ELISA was used to verify the expression of IL-7 and CCL21.Results Double digest and Positive cloning sequence comparison results showed that the target gene was inserted into the vector correctly.Real-time PCR and ELISA verified the expressions of IL-7 and CCL21 higher in Transfected B16F10 cell than in the wild type of B16F10 cell?P<0.05?.Adenovirus was successfully packaged and the titer of adenovirus vectors was 2.2x10¹¹pfu/ml.Conclusion Ad-IL-7-CCL21 dual gene adenovirus co-expression vector was successfully constructed,which can be successfully transfected and overexpressed in B16F10 cells,laying a foundation for further study of its effect on melanoma in vivo.PART2 Antitumor effects of Ad-IL-7-CCL21 adenovirus to mouse melanoma in vivoObjective To further explore the effect and possible mechanism of recombinant adenovirus Ad-IL-7-CCL21 in vivo.restructuring disease poison,the control group,low dose recombinant disease restructuring sick poison experimental intervention,measure and record the change of the back length to diameter of the transplanted tumor in mice,calculating volume of tumors,and draw the growth curve,measure the weight of tumors.HE staining was used to observe the cell morphology in tumor tissue sections of each group,and immunohistochemistry was used to compare the microvascular density and lymphocyte infiltration in sections of each group.Flow cytometry was used to detect the proportion of lymphocyte subsets in the spleen of mice to explore the anti-tumor mechanism.Methods B16F10 cells were subcutaneously inoculated in SPF C57BL/6 mice aged 6 weeks to establish a mouse model of melanoma transplantation.Divided the model rats into three groups randomly,respectively to the tumors had injection of high doseResults In vivo experiments showed that recombinant adenopathy venom could inhibit the growth of transplanted melanoma.The results of HE staining showed that the growth of tumor cells in the control group was significantly better than that in the other two groups.Immunohistochemistry showed that the lymphatic infiltration in tumor tissues in the experimental group was significantly higher than that in the control group,and the microvascular density in the high-dose group was significantly lower than that in the control group.The percentage of naive T cells in CD8+ T cells was significantly lower in the high dose group than in the control group?P<0.05?,and the percentage of effector T cells in CD4+ T cells was significantly higher in the high dose group than in the control group?P<0.05?.Conclusion The recombinant adenovirus Ad-IL-7-CCL21 can significantly inhibit the growth of transplanted tumor in vivo,which may be related to the improvement of T cell function,gathering of T cells and the inhibition of tumor angiogenesis.