Effect Of Porcine Indoleamine 2,3-Dioxygenase In Xenogeneic Cytolytic Activity Of Human Nature Killer Cells Against Porcine Endothelial Cells

Objective: To investigate the potential mechanisms of porcine indoleamine 2,3-dioxygenase(p IDO) gene regulation and the functions of p IDO in xenogeneic cytolytic activity of human NK cells against porcine Endothelial Cells.Methods: The 5?control region of the p IDO gene was obtained from Gen Bank and the potential regulation control sites were predicted using the bioinformatics technique. The p IDO promoter luciferase reporter gene vectors were constructed with different lengths of 5?control region, and were transfected into 293 cells. The luciferase intensity of transfected 293 cells extracting solution was tested. The critical regulation sequences were predicted through analysis the difference of luciferase intensity among reporter genes containing different length of p IDO gene 5?control region. Recombinant lentiviral vector p LVX-m CMV-Zs Green-PGK-Puro-IDO was constructed which contains the c DNA sequence of p IDO. Then the recombinant lentiviral vector was transfected into porcine endothelial cell line(PED) to rebuild a new cell line(PED-IDO) which was highly expressing p IDO gene. Then establish a co-culture system which contains transfected porcine endothelial cell and NK92 cell line. The heterogeneic Cytolytic Activity of NK92 cell line was assessed using flow cytometry and TUNEL method. The expression level of IFN-? and LFA-1 were assessed by ELISA and real-time PCR respectively.Results: there are 57 transcription factor biding sites located in the 2000 bp upstream of p IDO gene transcription start site. The recombinated vector p LVX-m CMV- Zs Green- PGKPuro-IDO was constructed and verified successfully by sequencing and double enzyme digestion. The result of luciferase reporter analysis shows that there are several regulation sequences in the upstream of the transcription start site, and-1035 ~-903 bp and-481 ~-378 bp are the two most critical regions of them. The p IDO gene high-expression cell line was constructed successfully. In the co-culture group, the expression levels of IFN-? and LFA-1 were down-regulated, and the cytolytic activity of NK92 cell was depressed.Conclusion: there are important control sites located in-481 ~-378 bp and-1035 ~-903 bp regions upstream of the transcription start site of p IDO. The Xenogeneic kill activity of NK92 cell line Against PED could be depressed by p IDO through inhibiting the expression of IFN-? and LFA-1.