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목적 : The chemokine receptor D6 and DARC (Duffy Antigen Receptor for Chemokines) are atypical receptors that act as a “scavenging receptor” for inflammatory CC and CXC chemokines. To modulate chemokine binding, we have previously generated proteins that mimic the D6 and DARC receptor. The purpose of this study was to newly develop more effective sulfated forms of D6 and DARC receptor and evaluate the effectiveness of chemokine trapping of the developed proteins in murine corneal allograft model.
방법 : Each extracellular domain of D6 and DARC receptor had 4 and 3 tyrosine sites that could be sulfated. We have mutated each of the extracellular tyrosine site and cloned the product with pET41a(+)vector.The recombinant plasmid was co-transformed with pSUPAR6-3SY-L3vector for tyrosine o-sulfation. We then used developed proteins to investigate chemokine-binding efficacy, immune cell activation in murine corneal allograft model by RT-PCR, flow cytometry and cell migration assay.
결과 : We successfully generated 5 kinds of sulfated D6 and 3 kinds of sulfated DARC receptors. These mimicking proteins were treated in the corneal allografts and allosensitization was effectively reduced in the mouse corneal allograft model. Flow cytometry data showed more decreased CD45+,CD19+,CD4+,CD11b+ cells and IFN-g,TNF-a,LYVE-1 and VEGFR fold changes significantly decreased in the cornea by sulfated D6 and DARC mimicking protein treatment compared to previous D6 and DARC receptor treated group. In the cell migration assay, migratory cells were drastically reduced by treating sulfated D6 or D
결론 : By successfully generating sulfated forms of D6 and DARC receptor proteins we showed better effectiveness in preventing allosensitization. These antagonists of the chemokine system represent a promising anti-inflammatory strategy in graft rejection.
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