The Research About Autoimmune Regulator (AIRE) On Effects Of Features And Functions Of Dendritic Cells Mediated By Adenovirus

Objective: To detect the infection efficiency of adenovirus to dendritic cells,establish the AIRE-DC vaccine and identify its features and biological functions.Methods:(1)Bone marrow cells were extracted from about 4 to 6 weeks old SD rats, after lyse RBC by red blood cells lysis, adherent cells were induced by cytokines, then we had mature rat bone-marrow derived dendritic cells(r BM-DCs) with high purity, observe the morphological changes of dendritic cells(DC) during the day;(2) Construct a recombinant AIRE gene adenovirus(Ad), modify DC with a specific gene by adenovirus infection,validate the ad efficiency of infection as well as gene modification by flow cytometry and western-blot method to suspending r BM-DCs and adherent DC2.4 cell line, identify the most suitable multiplicity of infection of adenovirus;(3) Extract the whole rat hepatoma cell line RH-35 antigen, loaded the successful AIRE gene modified DC with tumor antigen,detect m RNA expression levels of surface molecule CD83, CD86, CD11 c, and CD80, and MHC-II expression was detected by flow cytometry, search the influence for dendritic cell biology characters by AIRE gene.Results:(1)After various processing and cytokine inducing, bone marrow cell suspension comes to have high purity of DC cells, and they gradually increased, showed a significant elongated and slender dendritic form, and grow from the half-adherent status gradually transformed into adherent status;(2)Both infection efficiency of the adenovirus for DC2.4cell line and r BM-DCs are high, and may be modified on DC successfully, which DC2.4’s optimal MOI were 50 MOI, infection efficiency up to 90%; r BM-DCs’ optimal MOI were100 MOI, infection efficiency is about 70%, higher MOI leads to cytotoxicity;(3)r BM-DCs modified by the AIRE gene showed significant expression growth of various types of cell surface molecules after loading tumor antigens, CD80, CD83, CD11 c had a significant difference compared with the untreated group(p <0.05), CD86 and MHC-II molecules had a significant difference compared with the untreated group(p <0.01), while r BM-DCs surface molecules transfected with empty vector compared with the untreated group had no significant difference. And surface molecules expression of DC2.4 were both blow whether before or after infection and there was no significant difference.Conclusion:1. Adenovirus for primary DC and DC cell lines were infected with high efficiency,but higher infection efficiency for the cell line;2. Adenovirus optimal MOI of r BM-DCs is higher than DC2.4, adherent cells is more difficult to infect than suspension cells;3.AIRE can increase r BM-DCs surface molecules CD80, CD83, CD86, CD11 c and MHC-II class expression after antigen loaded, wherein the CD86 and MHC-II molecule expression was significantly increased.4.DC2.4 cell lines lack all kinds of cell surface molecules which DC should express,and after stimulation of tumor antigen the expression cells also has no significant changes,indicating the lose of normal biological function of DC after the normal cells was immortalized and taken too many times of passage.