Study On The Inhibitory Effect Of Active Immunotherapy On The Growth Of GL261 Glioma In C57BL Mice And Its Mechanism

Background:Glioma is known as a common and difficult to treat malignant tumor in the central nervous system tumors.Even with modern microneurosurgery technique,chemotherapy and radiotherapy,etc,the prognosis is still unsatisfied Which might relate to the invasion of glioma and the side effects of chemotherapy and radiotherapy.Hence,to improve the survival rate of patients with malignant glioma,a new treatment to remove the residual tumor cells after surgery is needed.As a special treatment,the active immunotherapy has been attracted by the researchers.Based on individualized immunotherapy,our laboratory developed a new specific active immunotherapy,using a new method to prepare the tumor vaccine.The new method not only remains the advantages of the old method,but also enhances the antigen stimulation/presentation as demonstrated by an enhanced anti-tumor effect in animal model.In this study,we used this new method to determine its effect on gliomaPart one The inhibitory effect of active immunotherapy on the growth of GL261 glioma in C57BL mice.Objective:To study the inhibitory effect of active immunotherapy on the growth of GL261 glioma in C57BL miceMethod:15 female C57BL/6 mice(8 weeks old)were randomizily divided into 3 groups:The first group:1ml of sterile saline,control group.The second group:old method:untreated tumor vaccine and immune adjuvant.The third group:new method:treated tumor vaccine and immune adjuvant.The mice were immunized with NS,or GL261 glioma cell lysate(old method)or GL261 glioma cells that have been treated with radiation,futher cultured for new oncogens expressed and then harvested as new vaccine.GL261 glioma cells(3 × 106/mouse)were inoculated into thing of each mouse.The inhibitory effect of immunotherapy on tumors was determined by the the tumor growth in the miceResults:Tumor growth:With new method,the growth of GL261 glioma was slowed down and the size of tumors were smaller than that of control group.(p=0.0052).In addition,the size of tumors of new method group were much smaller than that of old method group.(p=0.0347)Conclusion:The new method treatment had an inhibitory effect on the growth of GL261 glioma in C57BL/6 moue modelPart two Study on the inhibitory mechanism of active immunotherapy on the growth of GL261 glioma in C57BL mice.Objective:To study the inhibitory mechanism of active immunotherapy on the growth of GL261 glioma in C57BL miceMethod:15 female C57BL/6 mice(8 weeks old)were randomizily divided into 3 groups:The first group:1ml of sterile saline,control group.The second group:old method:untreated tumor vaccine and immune adjuvant.The third group:new method:treated tumor vaccine and immune adjuvant.The mice were immunized with NS,or GL261 glioma cell lysate(old method)or GL261 glioma cells that have been treated with radiation,futher cultured for new oncogens expressed and then harvested as new vaccine.The mechanisms by which the inhibitory effect was exerted were studied in several aspects:(1)ELISA for antibody titer for anti-GL261 glioma cells;(2)antigen-stimulated lymphocyte transformation assay;(3)antigen-stimulated lymphocyte killing assay;(4)active T subset analysis for CD4+IL-2+、CD8+、CD8+IL-2+cells with flow cytometryResults:Detection of peripheral blood immunological indices:(1)ELISA for GL261 antibody titer:compared to control group,GL261 antibody concentration increased significantly in mice immunized with treated GL261 antigen plus adjuvant.Compare the immunity group to the control group when the plasma dilution was 1:528000(p=0.012).(2)Lymphocyte transformation test:Compared to lymphocytes in peripheral blood in mice of the control group,PBMC were much bigger,lighter and rounder in mice of new method immunization group under the microscope.The results Brdu incoperation test indicated that there was significant difference of PMBC transformation and proliferation in peripheral blood of mice between new method immunization group and the control group(p=0.02).(3)LDH lymphocyte killing assay:LDH of the control group(253.6±50.043 IU/ml)was lower than that of the new method group(348.4±53.186 IU/ml).The difference was statistically significant by t test(p=0.0198).(4)immune cell clustering analysis by flow cytometry:With new method,the percentage of CD4+IL-2+cells could reach around 4%,which was higher than that of the old method group.The difference was significant(p=0.0286).With new method,the percentage of CD8+cells could reach around 46%,the percentage of CD8+IL-2+cells could reach around 14%,both were higher than that of the old method group(p=0.0102)Conclusion:The mechanism for active immunotherapy of anti-GL261 glioma effect might relate with the following aspects:(1)the active immunotherapy generated high titer(1:528000)of antibody specifically against GL261 glioma cells.(2)the active immunotherapy stimulated the lymphocyte in PBMC activation and proliferation;(3)the active immunotherapy triggered the proliferation of CD4+IL-2+cells,The increased CD4+IL-2+cells may have inhibitory effect on GL261 glioma.(4)the active immunotherapy also significantly increased the percentage of CD8+IL-2+cells,which may have inhibitory effect on GL261 glioma.