Expression of human decay-accelerating factor in porcine endothelial cells inhibits complement-mediated activation

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Abstract 605
Introduction In pig to primate discordant xenotransplantation, transgenic approaches of complement regulatory proteins (CRP) are feasible in controlling hyperacute rejection (HAR). Expression of human decay-accelerating factor (hDAF) render porcine endothelial cells (PEC) to resist the lysis of human complement. Activation of PEC by human complement plays a key role in HAR and acute vascular rejection (AVR). The objective of this study is to explore whether expression of hDAF on PEC can regulate human complement-mediated activation by evaluating expression of activation-related genes (IL-1α, E-selectin, and tissue factor) in transfected PEC treated with normal human serum (NHS).
Materials and methods Human DAF cDNA and elongation factor-1α (EF-1α) promoter were cloned from placenta and genomic DNA, respectively. We constructed pEFD mammalian expression plasmid. Transfection was performed by electroporation and neomycin-resistant clones were selected with G418.
Results Three stable expression clones were confirmed by Northern blot and immunofluorescence. They were treated with NHS to investigate the potential of anti-cytotoxicity, anti-coagulation and regulation of PEC activation. Our results showed that dead cell count (DCC) and lactate dehydrogenase (LDH) release of transfected PEC are less than that of the control (P < 0.01). Clotting time of the transfected PEC clones is prolonged significantly (P < 0.01). Expression of IL-1α, E-selectin, and tissue factor genes were analyzed in control and transfected PEC treated with NHS by detecting mRNA transcription. Our results showed that the expression of activation-related genes was down-regulated efficiently in transfected PEC as compared with control.
Conclusions Our results indicate that expression of hDAF in PEC can resist the cytotoxicity of human complement and possess anti-coagulant activity, and inhibit human complement-mediated activation.
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