异种移植排斥机理的探讨

An Approach to the Mechanism of Xenograft Rejection

补体的激活是超急性排斥的中心环节,为了研究经典及旁路途径在这种排斥中的作用,本研究建立了体外超急性排斥模型.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(methyl thagolyl tetragoliam,MTT)行补体依赖的细胞毒反应(complement-dependent cytotoxicity,CDC).人血清能溶解58±5%的猪血管内皮细胞.加入EGTA阻断经典途径后人血清的溶细胞率降为51±3%(P<0.01).同样Clq缺乏的人血清仅溶解37±7%猪血管内皮细胞(P<0.001).人血清50℃加热20min,阻断旁路途径后,其溶细胞率降为42±5%(P<0.001).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<0.001)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,血清的细胞毒作用恢复正常.同样,Clq缺乏人血清和B因子缺乏人血清分别加入Clq和B因子后,血清细胞毒作用亦恢复正常.在这一体外异种超急性排斥模型中,补体经典和旁路两条途径均参与超急性排斥反应.提示抑制猪/人之间的超急性排斥应考虑补体两条途径均激活的问题.

Complement activation is central to the rejection of discordant xenografts. In order to asses the respective roles of direct and alternative paths, an in vitro model of hyperacute rejection in the pig-to-human donor - recipient combination was designed, using a complement - dependent 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 direct pathway by using Clq- deficient human sera, only 37±7% of pig endothelial cells were killed (P< 0.001 versus normal serum). When the alternative pathway was selectively inhibited by factor B-deficient human serum, only 42±10% of porcine endothelial cells (P<0.001versus normal serum) were lysed. Syngenic normal pig serum and heat - inactivated serum were not cytotoxic. Mixing serum of direct pathway defect with serum of alternative pathway defect restored the cytotoxicity to normal levels. Similarly, the cytotoxic activity of deficient serum supplemented with purified Clq and 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 human's should take into account the dual activation of complement in this donor -recipient combination.