Investigation Of Defective Interfering Particles Of Paramyxovirus,Tianjin Strain On Apoptosis In Rat Neurogliocytoma

Objective Defective interfering(DI)particles are virus with subgenome,which are generated from the wrong replication.Compared with the standard virus,the DI particles are more secure for which can not perform a complete copy process and not independent replication.Paramyxovirus,Tianjin Strain was isolated from the lungs of cotton-eared marmosets which were burst out to catch the acute upper respiratory infection in 1999.It was identified being a member of the Respirovirus of Paramyxovirus family,and a new genotype of SeV which has the nearest genetic relationship with it.Respecting the hypopathogenicity of Sendai Virus for human,meanwhile some Sendai Virus genetypes had been confirmed that had inhibitory action to timor,this thesis will search on the apoptosis of Sendai Virus defective interfering particle to tumor,and explore the treatment of tumor via SeV DI particles without exogenous gene expression and dissolveing the tumor nor adjunctive therapy.By means of the mechanism of treatment on tumor,a new way is provided for eradicating cancer.Methods The Paramyxovirus,Tianjin Strain was obtained by cultivation in the chick embryo.The DI particles of Paramyxovirus,Tianjin Strain were obtained through ultracentrifugation and sucrose density gradient purification.The Paramyxovirus,Tianjin Strain was inactivated by ultraviolet irradiation.The DI particles and the inactivated Paramyxovirus,Tianjin Strain were identified by showing under the electron microscope,observing the CPE after inactivated virus particles being inoculated in rat neurospongioma cell,C6 strain and detecting the HA titers of allantoic fluid collected after cultivating DI particles and inactivated virus particles in the chick embryo.The rat neurospongioma cell,C6 strain were chltured in vitro.The DI particles were inoculated in rat neurospongioma cell,C6 strain at different titer,besides we set groups of Paramyxovirus,Tianjin Strain virus particles,inactivated virus particles,and normal saline as control.At different time-points(12,24,48 and 72 h)the changes in cell morphology was observed by hematoxylin-eosin(HE)staining,apoptosis was detected by agrose gel electrophorsis of DNA fragments and TUNEL kit,Annexin V FITC plus PI staining for apoptotic and necrotic rate was examined by fiow cytometer.Rat neurospongioma cell,C6 strain were inoculated subcutaneously in rat to make the bearing cancer model,then the DI particles were injected to the cancer model,besides we set groups of Paramyxovirus,Tianjin Strain virus particles,inactivated virus particles,and normal saline as control.To study the apoptosis of DI particles in Rat neurospongioma cell by observing the growth changes of subcutaneous tumor by measuring the diameter of tumor,and Isolating the tumor from subcutaneous to observe the histopathological changes with pathological section staining and detect the apoptosis of tumor cells with TUNEL kit.Results It was showed that the formulations isolated by purification were DI particles which were different from the standard virus,the virus particles had no ability of replication and infection after being inactivated by ultraviolet irradiation.In vitro,the results showed that the groups of Paramyxovirus and DI particles had observed the morphological changes of tumor cells by hematoxylin-eosin(HE)staining,DNA ladder was detected in some samples,Apoptotic cells were found by flow cytometry and TUNEL assay,and apoptotic rate in different titer of DI particles was significantly higher than in normal saline control group(P<0.05).Apoptotic rate in the group of inactivated virus particles were no more than in normal saline control group(P>0.05).In vivo,DI particles can also inhibit the growth of the tumors compared with the saline control group,and the result of hematoxylin-eosin(HE)staining show that the subcutaneous tumor had fewer tumor cells in the DI particles group.Apoptotic cells were found by TUNEL assay,the rate of apoptosis was increased after the DI particles being injected to the cancer model.Conclusion The DI particles of Paramyxovirus,Tianjin Strain can induce apoptosis in Rat neurospongioma cell in vitro and in vivo,which shows a time and dose-dependent manner.This study showed that the DI particles of Paramyxovirus,Tianjin Strain have the potential capability to neurogliocytoma treatment,and a new way will be provide for eradicating cancer.