| Objective: To culture the rat bone marrow derived immature dendritic cell (DC) and transfectionof it with recombinant adenoviral vector expressing rats CCR7gene；To study the role of imDCwhich transfected recombinant adenovirus chemokine receptor7(CCR7) on immune tolerance inrat penetrating keratoplasty (PKP) in high-risk eyes and to investigate the mechanism of immunehyporesponsiveness induced by it.Methods: Bone marrow-derived imDC of wistar rats were cultured with recombinant ratgranulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin (IL)-4. Transfectionof imDC with a recombinant adenoviral vector expressing rats CCR7gene, then analyzed them byscanning electron microscopy and flow cytometry. Used Immunofluorescence assay to analyz theexpression of CCR7protein on it. Thirty Vistar rats and60SD rats were respectively used asdonor and recipient. Corneal neovascularization was induced by alkaline burn in the central corneaof recipient rat. The recipients were randomly divided into control, imDC, adenovirus void vectortransfected (imDC+Ad) and recombinant adenovirus CCR7transfected (imDC+Ad-CCR7) groups,fifteen cases in each group. One week before operation and three days after operation, SD ratswere injected with0.1ml PBS, donor bone marrow-derived imDC of1×107, donor bone marrow-derived imDC transfected with adenovirus void vector of1×107, and donor bone marrow-derivedimDC transfected with recombinant adenovirus CCR7of1×107via tail vein, respectively. Thesurvival condition of corneal grafts was observed and scored by slit lamp at each day afteroperation. In each group, six recipients were executed on the14th day after transplantation. Thecorneal grafts were stained by hematoxylin-eosin (HE). PT-PCR was used to detect the mRNAexpression of Th1-type cytokines IL-2, interferon (IFN)-γ and Th2-type cytokines IL-4, IL-10.Result: The DC were validated and identified through microscopy, scanning electron microscopyand flow cytometry analysis. By immunofluorescence assay, significant CCR7protein expressionwas detected in CCR7gene-transfected imDC, but not in imDC and the void adenoviral vectorstransfected imDC. The median survival time (MST) of imDC+Ad-CCR7group was signifyca-ntly longer than of control, imDC and imDC+Ad groups (all P<0.01). The expression level ofmRNA on Th1-type cytokines IL-2and IFN-γ of imDC+Ad-CCR7group was significantly lowerthan that in the control, imDC and imDC+Ad groups; the expression level of mRNA on Th2-typecytokines IL-4and IL-10of imDC+Ad-CCR7group was significantly higher than in the control,imDC and imDC+Ad groups (all P<0.05).Conclusion: The CCR7gene was successfully transfected into imDC and expressed CCR7protein. The imDC which transfected recombinant adenovirus CCR7is an effective treatment ininducing immune hyporesponsiveness in rat PKP. The mechanism of immune tolerance inducedby it might be influence the balance of Th1/Th2cytokines. |
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