Bioluminescence Imaging Of Distribution And Homing Of Labled DCs In Vivo

Interferon and nucleotide analogues are conventional drugs for the treatment of hepatitis B virus(HBV) infection in the clinic, however hepatitis B virus is rarely eliminated and some of the patients have no response, so new treatment strategy is greatly needed. The main reason for the persistence of HBV is the lack of HBV specific antigen presenting cell, which makes T cell's cytotoxicity abnormal, and could not clear the virus efficiently. Dendritic cells(DCs) is the professional antigen presenting cell in the peripheral blood and lymphoid tissue, involved in the maintenance of T-cell-mediated immune response. These capacities make DCs a key cell population for adoptive immunotherapy. While early clinical trials clearly show the potency of DC therapy, there are still poor understanding about the function of DCs in the clearance of HBV, preventing its introduction as a standard treatment. So, studying the distribution and function of DCs in disease mice models can accelerate the standardization of treatment process. In this study,we label isolated DCs with Gaussia luciferase in vitro, and then they were injected into Fluc labeled HBV mouse model. Using dual bioluminescenece imaging, we have done a preliminary study about the distribution and migration of DCs in disease animal models. The methods and results are as follows:1. With IRES mediated membrane-bound Gluc and Fluc as reporter genes respectively, we constructed the adenovirus shuttle vectors and control vectors which containing HBc fragment, AD-HBc, AD-Fluc, AD-Fluc-HBc, AD-Gluc, AD-HBc-Gluc; then transfection into bacteria, after recombination, adenovirus vectors were packaged in 293 cells and we got recombinant adenovirus. After amplification and purification, Hepa1-6 was infected with recombinant adenovirus , more than 95% was infected; the level of luminous intensity of 105 live cells of Gluc can still to 107 RLU in vitro. Gluc luminous intensity was higher than Fluc by two orders of magnitude through in vitro determination of luciferase activity. 2. Mice were hydrodynamic transfected with plasmid pUMVC3-Flt3L twice on day 1 and day 6, changes in spleen and liver were observed on day 12, spleen lymphocytes were isolated, and DCs were sorted with CD11c magnetic microbeads. flow cytometry detected the purity of dendritic cells, and analyzed the phenotype of dendritic cells. The results demonstrated that after twice hydrodynamic transfection of plasmid pUMVC3-Flt3L, the weight of the spleen was significantly higher than the control mice, about 4-5 times, CD11c + cells was about 30% of the spleen lymphocytes. Each mouse isolated dendritic cells can be to 5×107. The purity of separated dendritic cells was more than 96%, besides the phenotype of separated dendritic cell had not changed, so the method didn't affect the mature of dendritic cells.3. Recombinant adenovirus AD-Gluc and AD-HBc-Gluc infect CD11c + dendritic cells at MOI 200: 1, in vitro detection the infection efficiency and the level of the expression of Gluc in living cells, the in vivo distribution of intravenous and subcutaneous injected DCs was observed by bioluminescence imaging, and through dual bioluminiscence imaging, homing of Gluc labeled dendritic cells was observed in Fluc labeled HBV mice in vivo. The results show that recombinant adenovirus's infection efficiency to CD11c + dendritic cells was about 50%, and the sensitivity of 106 infected CD11c + dendritic cells was 8×107 RLU. Because the luminescence of Gluc can decay very fast, convenient to Flu imaging immediately in vivo, it can be easily to establish Gluc and Fluc dual bioluminescence system, it provide a good animal model platform for the study of the interaction between HBV and the host.In conclusion, this study use recombinant adenovirus method, and Gluc as a reporter gene to label in vivo separated dendrite cell, inject to the Fluc labeled HBV mouse model, combined in vivo dual bioluminescence imaging to establish a luciferase mouse model. This model can be real-time monitoring of distribution, migration and function of dendritic cells, provide a visualized, sensitively and convenient animal model system to evaluate of clinical vaccine efficacy of dendritic.