Experimental Research Of Recombinant Vaccinia Virus RVV-CCL5-SSTR2-Luc+in Targeted Cancer Treatment In Vitro

In recent years,tumor immunotherapy has achieved great success as another important treatment after surgery,radiotherapy and chemotherapy and targeted therapy,and has gradually become one of the standard treatment methods for cancer.Oncolytic virus-mediated anti-tumor immunotherapy has achieved remarkable results and represents a new cancer immunotherapy.At present,the commonly used oncolytic viruses include vaccinia virus,adenovirus,herpes virus,etc.,and the research group often genetically modify vaccinia virus because of its high safety,large genomic capacity,high replication efficiency,rapid spread,clear side effects and clear treatment.The expression product of the vaccinia virus TK gene can utilize the deoxyuridine analog Brdu to block the synthesis of viral nucleic acid,inhibit the replication of vaccinia virus in the cell,and then screen out the vaccinia virus with TK gene deletion.CCL5 and SSTR2 have strong anti-tumor effects.CCL5 is a member of the chemotactic cytokine CC subfamily,it plays an important role in the occurrence,development and outcome of tumors.SSTR is a somatostatin receptor,in which the type 2 receptor SSTR2 is considered to be most closely related to tumors.SSTR2 can inhibit tumor proliferation,induce tumor cell apoptosis,and participate in immune system regulation.In the previous work,our group has successfully constructed the poxvirus eukaryotic expression vector pSC65-CCL5-SSTR2-Luc and its control pSC65-Luc,which are recombinant human CCL5 and SSTR2 genes,and obtained recombinant oncolytic pox virus by homologous recombination.The control group rVV-Luc+ has completed the screening work,and the rVV-CCL5-SSTR2-Luc+ has completed the first 10 rounds of screening.The study continued on this basis,and a total of 23 rounds were screened,and the WB verification was performed by rVV-CCL5-SSTR2.-Luc+ infected tumor cells correctly expressed CCL5 and SSTR2 proteins,confirming that the recombinant poxvirus rVV-CCL5-SSTR2-Luc+ which correctly expressed CCL5 and SSTR2 was successfully screened and purified.Analysis of the relationship between viral plaque and virulence confirmed that the TK gene deletion of rVV-CCL5-SSTR2-Luc+ contributes to the safety of poxvirus.The killing activity of rVV-CCL5-SSTR2-Luc+ on tumor cells was evaluated by CCK8 assay.The results showed that rVV-CCL5-SSTR2-Luc+ can inhibit the activity of HCT116,HELA and HCCLM3 cells,and its inhibitory effect on tumor cells The increase in infection time is gradually increasing.Through the study of 10 potential effector targets for the oncolytic effect of recombinant oncolytic virus rVV-CCL5-SSTR2-Luc+,the mechanism of its oncolysis effect was discussed.The results showed that in most tumor cells,the oncolytic pox virus may promote the apoptosis of tumor cells by stimulating the expression of the proapoptotic protease caspase-3 and the apoptosis-promoting factor Bax.In some tumor cells,the expression of anti-apoptotic factors survivin and Bcl2 is up-regulated by stress.In some tumor cells,the oncolytic poxvirus rVV-CCL5-SSTR2-Luc+ can block the formation of tumor endothelial blood vessels by decreasing the expression of VEGFA protein,thereby exerting anti-tumor effect.This study laid a solid experimental and theoretical basis for the further in vivo and clinical research of rVV-CCL5-SSTR2-Luc+ in the treatment of tumors.Research results:1.The recombinant poxvirus rVV-CCL5-SSTR2-Luc+,which correctly expressed CCL5 and SSTR2 proteins,were successfully screened.2.The analysis of the relationship between viral plaque and recombinant poxvirus virulence showed that the pathogenicity of rVV-CCL5-SSTR2-Luc+ was lower than that of wild-type poxvirus WR,confirming rVV-CCL5-SSTR2-The deletion of the TK gene of Luc+ contributes to the safety of the poxvirus.The effect of rVV-CCL5-SSTR2-Luc+ on tumor treatment in vitro was evaluated.The results showed that rVVCCL5-SSTR2-Luc+ inhibited the cell viability of colorectal cancer cell line HCT116,cervical cancer cell line HELA,and liver cancer cell line HCCLM3.And the inhibitory effect of rVV-CCL5-SSTR2-Luc+ on cells is gradually increased with the increase of infection time.3.To evaluate the efficacy of recombinant oncolytic virus rVV-CCL5-SSTR2-Luc+ in the treatment of tumor in vitro and to explore its mechanism of action.In most tumor cells,the oncolytic pox virus may promote tumor cell apoptosis by stimulating the expression of the proapoptotic protease caspase-3 and the apoptosis-promoting factor Bax.In some tumor cells,the expression of survivin and Bcl2 was up-regulated by stress.In some tumor cells,the oncolytic poxvirus rVV-CCL5-SSTR2-Luc+ can block tumor angiogenesis by reducing the expression of VEGFA protein,thereby exerting anti-tumor effect.