Effect of human CRPs and Bcl-xl combined transgenes on protection of porcine endothelial cells

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Abstract 614
Introduction In this study we investigated, respectively, the effect of expression of double human complement regulatory proteins (CRPs) and double human decay accelerating factor (hDAF) and Bcl-xl in porcine endothelial cells (PEC) on prevention of PEC activation by human complement.
Materials and methods Using PEC in vitro culture model, human membrane cofactor protein (MCP), CD59 and Bcl-xl expression plasmids were transfected into hDAF-PEC, respectively. The death rates of PEC caused by human complement, and the levels of E-selectin expression stimulated with recombinant human TNF-α were analyzed.
Results In different concentration of human serum 1:1, 1:2 and 1:4, the death rates of hDAF-PEC transfected with hMCP were 18 ± 4%, 10 ± 3%, 4 ± 1%, respectively, and that of hDAF-PEC transfected with hCD59 were 15 ± 3%, 8 + 2% and 4 ± 2%. Both were significantly lower compared with that of simple hDAF-PEC (30 ± 5%, 18 ± 4%, 10 ± 3%) (P < 0.05). But flow cytometry displayed similar expression level of E-selection. The death rates of hDAF-PEC transfected with Bcl-xl expression plasmid were 25 ± 6% (1:1), 15 ± 4% (1:2) and 10 ± 2% (1:4), and similar to that of simple hDAF-PECs (P > 0.05). The levels of E-selectin and hTNF-α expression in hDAF-PEC tansfected with Bcl-x1 group were significantly lower compared with that of simple hDAF-PEC (P < 0.05) and unmodified PEC (P < 0.01). Immunohistochemistry examination disclosed that transgene of human DAF could reduce apoptosis of EC, which could be improved by further transfection of Bcl-xl plasmid.
Conclusions 1. The results indicated that further transfection of hMCP or hCD59 could provide stronger protection to hDAF-PEC, but none of hDAF, hMCP or hCD59 gene could be of benefit to the prevention of EC activation. 2. Bcl-xl was not useful to prevent complement activation, but was helpful to reduced E-selectin expression. 3. Human DAF may not resist the expression of E-selection, but may reduce complement activation. 4. Moreover, the presence of hDAF and Bcl-xl expressed in double transgenic PEC could benefit to reduce EC apoptosis and inhibit complement activation which to prevent xenograft rejection.
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