| Malignant tumor is a kind of disease that seriously endangers human health,the newest finding published by International Agency for Research on Cancer indicates that,there would be about 18 million new cases of cancer worldwide,and 9 million patients would die of it,standing the scond seriousest noninfection disease among humans.The surgery,radiation and chemotherapy are the cancer main treatment methods.These traditional treatments can improve the entirety survival rate,while some patients cannot have satisfactory results by those therapies,and the recurrence and metastasis of tumor cannot be controlled by those conventional options,and they always lead to the drug resistance with little noxious effect.Immunotherapy,targeted therapy and oncolytic virus may become a novel anti-tumor strategy,which provide new hope for conquering cancer.The fields of oncolytic viruses are growing areas in identifying new therapied for cancer treatment,oncolytic virus specifically infects tumor cells and lyses them,it is safe,efficient and has few side effects.Nowadays there are many oncolytic virus entering the clinical trials or converting into drugs on the market.Influenza viruses are single-stranded,negative,segmented RNA viruses,science firstly be reported in 1904 said that the influenza viruses may be beneficial for alleviating leukaemia,several other studies have also demonstrated the ability of influenza viruses to kill tumors.At present using reverse genetics the wild-type viruses can be modified to be a safe and effective biological reagent for tumor treatment.The purpose of the research is to evaluate the oncolytic activity of various wild influenza viruses to screen out one strain with better tumor preference,and then to modify the gene of the virus to improve its safety and efficacy.For this purpose,the following works were carried out.Firsty,we establisded a cell platform in vitro for evaluating cell killing ability of oncolytic viruses by immunofluorescence and CCK-8 kit,then the oncolytic effect of different types of wild influenza virus were evaluated by a panel of cell lines which consisting of 7 different cancer cells.Among them,three strains show better preference to the majority of the carcinomas.Then we detect the antibody titers specific for influenza virus in the serum of people in different ages,and we also detect the replication ability of the viruses,finally we decided to use A/WSN/1933 in the following study.Then we focus on the modification work of virus.On the one hand,in order to enhance the specific selective killing of virus to tumor cells,we try to motify the NS1 gene of A/VSN/1933,on the other hand,in order to improve the oncolytic capacity we attempt to make a transformation on the HA gene of A/WSN/1933.Finally,we successfully rescued a virus named WSN-V1194HA-73NS1 by using reverse genetics.The oncolytic capacity of the virus was evaluated on cancer cells and in models of tumor bearing mice.It was revealed that the efficacy of WSN-V1194HA-73NS1 was better than A/WSN/1933.Finally,the oncolytic mechamism of WSN-V1194HA-73NS1 was investigated by methods of Immuno-histochemistry and flow cytometry.It was concluded that WSN-V1194HA-73NS1 can induce apoptosis of cancer cells and T cell proliferation and activation in tumor immune microenvironment.However,the more detailed mechanisms still need to be futher investigated.Above all,we successfully obtained a safe and effective oncolytic virus WSN-V1194HA-73NS1,and the efficacy of WSN-V1194HA-73NS1 was evalusted both in vitro and vivo,using a series of cancer cell lines consisting of 32 diflferent cells and 4 different mice xenograft models.In addition,the mechamism of oncolytic therapy was also preliminarily explored.The study laied a solid foundation for developing a variety of novel oncolytic viruses for tumor treatment in the future. |
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