人补体对猪血管内皮细胞的溶细胞效应研究

An approch of the cytotoxicity of human complement to porcine endothelial cells

选择猪血管内皮细胞为靶，人血清为天然抗体和补体源，用四唑盐法（ＭＴＴ）行补体依赖的细胞毒反应（ＣＤＣ），建立了体外超急性排斥模型。人血清能溶解５８±５％的猪血管内皮细胞。Ｃ１ｑ缺乏的人血清（阻断经典途径）仅溶解３７±７％猪血管内皮细胞（Ｐ＜０．０１）。Ｂ因子缺乏的人血清（阻断旁路途径）仅溶４２±１０％的猪血管内皮细胞（Ｐ＜０．０１）。同种猪血清及加热灭活补体的人血清不溶猪血管内皮细胞。将经典途径及旁路途径缺陷的人血清等体积混合，其细胞毒作用恢复正常。同样，Ｃ１ｑ缺乏的人血清和Ｂ因子缺乏的人血清分别补加Ｃ１ｑ和Ｂ因子后，血清细胞毒作用亦恢复正常。因此，补体经典和旁路两条途径可能均参与体内超急性排斥反应。提示抑制猪／人之间的超急性排斥应考虑补体旁路途径激活的问题。

Complement activation is central to the rejection of discordant xenografts.In order to assess the respective roles of classical and alternative pathways of complement activation,an in vitro model of hyperacute rejection in the pig to human donorrecipient combination was designed,using a complementdependent cytotoxicity test with human serum as the source of xenogenic antibodies and complement.The cytotoxic activity of the sera was evaluated by a colorimetric assay using MTT. Pure human serum lysed 58±5% of pig endothelial cells.Selective inhibition of the classical pathway by using C1q deficient human sera,only 37±7% of pig endothelial cells were killed( P <0 01 versus normal serum).When the alternative pathway was selectively inhibited by using factor B deficient human serum which could only lyse 42±10% of porcine endothelial cells( P <0 01 versus normal serum).Syngeneic normal pig seurm and heat inactivated serum were not cytotoxic. Mixing serum with deficient classical pathway and serum with deficient alternative pathway restored the cytotoxicity to normal levels.Similarly,the cytotoxic activity of deficient serum supplemented with purified C1q of factor B at physiological concentrations reached that of normal human serum.In this model of in vitro hyperacute rejection,both pathways of complement activation were involved,suggesting that regimens designed to inhibit hyperacute rejection of swine xenografts into humans should take into account the activation of alternative complement pathway.