Effction Of Dendritic Cells Modified By The Recombinant Adenovirus Of CTLA4Ig And α4β7 In Treating Food Allergy

PARTⅠTRANSDUCT ADCTLA4IG AND ADα4β7 INTO DENDRITIC CELLSObjective To identify the infected efficiency of AdCTLA4Ig and Adα4β7 by amplifying AdCTLA4Ig and Adα4β7.To construct the dendritic cell vaccines transducted by AdCTLA4Ig and Adα4β7.Methods AdCTLA4Ig and Adα4β7 were transfected to 293 cells. The virus titer were detected by TCID50.The bone marrow cells were collected from the tibial and femurs of the mice,and cultured with GM-CSF and IL-4 For 7 days.The morphology of the cells were observed by light microscope.The dendritic cells were identified by FCM and infected with AdCTLA4Ig and Adα4β7.The expression of purpose gene were detected by fluorescence microscope.The phenotypic expression of DC in different stages were identified by FCM.Result The virus titer of AdCTLA4Ig and Adα4β7 were 5.01×10~8pfu/ml and 4.8×10~8pfu/ml.42%of the dendritic cells were showed the Phenotype of CD11c which were definited as the dendritic cells.About 80%of DC coexpressed CTLA4Ig andα4β7 by fluorescence microscope. The infected efficiency was the highest when MOI was 50.The expression of costimulatory molecule CD86 on the DC was decreased compared to normal matured DC,but the expression of MHCⅡwasn't changed.Conclusion The dendritic cells could be transducted efficiently and have the phenotypes of tolerant dendritic cell.The expression ot costimulatory molecule CD86 on the tolerant DC was decreased compared to normal matured DC,but the expression of MHCⅡwasn't changed.PARTⅡTHE STUDY ON THE PREVENTION AND TREATMENT OF FOOD ALLERGY BY TOLERANT DCSObjective To investigate the preventative and therapeutic effects of AdCTLA4Ig/Adα4β7-DC in the ovalbulmin-induced food allergy BALB/c mice.Methods Female BALB/c mice aged at 4-6 weeks fed on the ovalbulmin-free diet were randomly divided into 6 groups,including prevention group(group P) administered tolerant DC after being basic sensitized,treatment group(group T) administered tolerant DC after being challenged with OVA.Both the prevention group and treatment group were divided into P1,P2 and T1,T2 according to the administration of AdCTLA4IgDC or AdCTLA4Ig/Adα4β7-DC.At the same time,the normal mice sensitized ande challenged with normal saline were set as the negative control.Meanwhile,the mice sensitized and challenged with OVA were set as the positive control.(1) The symptoms were determined.(2) The tissues of small intestine were histologically examined by both HE and toludine blue stain.(3)The level of the OVA-specific IgE in serum were measured by ELISA.(4) The level of IL-10 in murine intestine were analyzed by immunohistochemical methods.(5)The expression of TGF-βandα4β7 in intstine were determined by immunofluorescent.Result Compared to the mice of positive group,intravenous injecting with AdCTLA4IgDC or AdCTLA4Ig/Adα4β7-DC in group T1 and T2 decreased diarrhea accurrence,reduced the level of the OVA-specific IgE in the serum,inhibited the significant inflammatory reactions,such as the accumulation of the lymphocytes and plasmacytes in lamina propria(LP) and the focal necrosis and shedding of the epithelial cells in intestinal villi,the accumulated and degranulated mast cells in LP, increased the expression of the IL-10 in intestine.Compared to the positive group,there was no significant improvement in group P1 and P2.The was no difference in the expression of every group.The level ofα4β7 in the murine intestine of group T2 was highest.Conclusion This study demonstrated that intravenous injecting with AdCTLA4IgDC or AdTLA4Ig/Adα4β7-DC may be of potential research value in the treatment of food allergy.It was able to relieve the acute episode,inhibit the inflammatory reactions,promote the establishment of immune tolerance.Intravenous injecting with tolerant DC had no preventative effect of food allergy.