The Anti-tumor Effect Of Gene-oncolytic Adenovirus SG635-SF To Ovarian Tumor

Background:In recent years,ovarian cancer incidence is increasing in China. Due to the lack of typical symptoms and early diagnosis,ovarian cancer has been commonly diagnosed in the late. At present,the treatments for cancer is very limited. Surgery,chemotherapy,radiotherapy effect is not ideal. Therefore,we need to find a new type of ovarian cancer treatment immediately. At present,immunotherapy can play an important role in tumor treatment. Using oncolytic adenoviruses can improve the immune against tumor cells.Oncolytic adenoviruses is engineered, can specific infection in tumor cells and can complete the self replication in tumor cells, causes tumor cracking. The virus lack necessary replication gene in normal cell, so do not produce damage in normal cell.CD47 is a kind of cell surface molecules, it is a member of the immunoglobulin(Ig) superfamily, that binds several proteins including integrins and thrombospondin-1, and has been implicated in diverse physiologic processes including cell migration, T cell and dendritic cell(DC) activation, and axon development. CD47 can high expression in many tumor cell surface, its mechanism is combined with macrophage surface SIRP alpha, release the "don’t eat me" signal to escape the phagocytosis of macrophage, achieve the goal of immune escape.Signal regulatory protein alpha(SIRP alpha) is inhibitory receptors, SIRP alpha expression relatively restricted to bone marrow cells, such as macrophages, neutrophils, dendritic cells, etc. SIRP alpha is one of the members of membrane proteins can mediate white blood cells function. CD47 is its ligand, they constitute SIRP alpha-CD47 way. In SIRP alpha-CD47 way, SIRP alpha on the surface macrophages combination with CD47 on tumor cell surface, recognition of tumor cell as "self" cells, thereby inhibiting the phagocytosis of macrophage. SIRP alpha-CD47 way can also help macrophages identify normal cells and aging cells, make aging cells are cleared.Result:In this study, we use oncolytic adenovirus SG635-SF carrying the SIRP alpha(CV1)-Ig G1 Fc gene segment observe the treatment effects in human ovarian cancer cells in vitro model and mice ovarian cancer transplantation tumor in vivo model. The main results are the following in six aspects.1、Construction and identification of oncolytic adenovirus SG635-SF. We constructed type 35 oncolytic adenovirus SG635-SF via technology of molecular cloning. PCR results confirmed the existence of desired genes SF, while absent of wild viruses.2、Expression of genes SF. Results of Western Blotting assay confirmed the expression of fusion protein SF by oncolytic adenovirus SG635-SF. ELISA assay showed that SG635-SF could effectively express SF.3、Fusion protein SF could specifically bind to CD47 and close it. Oncolytic adenovirus SG635-SF infected HO8910 and SK-OV-3,Results of indirect immunofluorescence assay and Flow cytometry confirmed that fusion protein SF could specifically bind to CD47 and close it.4、Oncolytic adenovirus SG635-SF could replicate in ovarian cancer cell line and have killing effect. Results of virus proliferation assay showed that the oncolytic adenovirus SG635-SF and could replicate in ovarian cell line SK-OV-3 and HO8910, at the peak volume of replication is about 1,000 times. The results of MTT showed SG635-SF had a better killing effect than SG635 in ovarian cancer cell line5、Oncolytic adenovirus SG635-SF could induce human ovarian cancer cells c HO8910 and SK-OV-3 apoptosis. Used proliferative SG635, SG635- SF, non-proliferative Ad-SF,Ad-blank infect HO8910,found proliferative SG635-SF can effectively cause the cell apoptosis. The results showed closed CD47 can induce the cell apoptosis.6、Oncolytic Adenovirus SG635-SF and its control virus could obviously inhibit the growth of ovarian cancer in vivo.Treatment nude mouse model of ovarian cancer by using SG635-SF and its control virus, results showed that oncolytic adenoviruses could inhibit the growth of tomor. The effect of SG635-SF was better. Conclusion Our results showed that oncolytic adenovirus SG635-SF could effectively infect ovarian cancer cell line SK-OV-3、HO8910 and mediate the expression of exogenous genes.In vitro, SG635-SF promoting the apoptosis of ovarian tumor cells, inhibiting cell growth; In vivo, SG635-SF inhibiting the growth of ovarian tumor cells and activating the immunological reaction effectively. The new strategy could be targeting tumor stem cells treatment.