异种肝移植术后组织因子激活介导受体凝血功能障碍的机制

Mechanism of coagulation dysfunction in the recipients mediated by tissue factor activation after liver xenotransplantation

目的探讨异种肝移植术后导致受体凝血功能障碍的组织因子(TF)激活机制。方法实施3例α-1,3-半乳糖基转移酶基因敲除(GTKO)小型猪-藏酋猴异种脾窝异位辅助性肝移植术。观察受体术后凝血功能变化情况。采用逆转录聚合酶链反应(RT-PCR)及免疫组织化学染色检测移植术前及术后不同时间点受体原肝和移植肝组织的猴TF及猪TF信使核糖核酸(mRNA)和蛋白的表达情况。检测正常对照猴、移植术前受体猴及移植术后2 h受体猴的外周血单核细胞(PBMC)的复钙时间以评价受体凝血状态。结果 3例受体在术后均出现了不同程度的凝血功能障碍,表现为纤维蛋白原水平降低、血小板计数减少。猴TF蛋白在术后受体原肝中呈阳性表达,在术前及术后的移植肝中呈阴性表达;而猪TF蛋白在原肝和移植肝中始终为阴性表达。术后2 h,猴TF mRNA在原肝中较术前上调(2.10±0.24)倍,而猪TF在移植肝中较术前上调(1.42±0.15)倍,两者比较差异有统计学意义(P=0.014)。与正常对照猴PBMC和移植术前受体猴PBMC比较,术后2 h受体猴PBMC复钙时间明显缩短,差异均有统计学意义(均为P<0.001)。结论在异种肝移植术后凝血功能障碍发生时,受体原肝TF激活程度显著高于移植肝TF。受体原肝TF激活是导致异种肝移植术后凝血功能障碍的主要原因。

Objective To investigate the mechanism underlying the activation of tissue factor（TF） that leads to coagulation dysfunction in the recipients after liver xenotransplantation. Methods Auxiliary heterotopic liver xenotransplantation was performed in 3 minipigs with α-1,3-galactosyltransferase gene-knockout（GTKO） as the donors and Tibetan macaque（Macaca thibetana） as the recipients. Postoperative coagulation function changes in the recipients were observed. Reverse transcription polymerase chain reaction（RT-PCR） and immunohistochemical staining were adopted to quantitatively measure the expression levels of monkey and minipig TF messenger RNA（mRNA） and protein in the liver tissues of the primary and transplant livers at different time points before and after transplantation.The recalcification time of peripheral blood mononuclear cell（PBMC） was recorded in the normal control monkeys and the recipient monkeys before and 2 h after liver transplantation to evaluate the coagulation status in the recipients.Results All three recipients presented with different degrees of coagulation dysfunction after surgery, manifested as a decrease in fibrinogen level and a reduction in platelet count. The monkey TF protein was positively expressed in the primary livers after surgery, whereas negatively expressed in transplant livers before and after liver transplantation.The minipig TF protein was negatively expressed in both primary livers and transplant livers. At postoperative 2 h,monkey TF mRNA was up-regulated by（2.10±0.24） times in the primary liver compared with the preoperative level,whereas the minipig TF mRNA was up-regulated by（1.42±0.15） times compared with preoperative level. There was statistical significance between the primary livers and transplant livers（P=0.014）. Compared with PBMC in the normal control monkeys and recipient monkeys before liver transplantation, the recalcification time of the PBMC in the recipient monkeys was significantly shortened at postoperative 2 h（both P〈0.001）. Conclusions At the presence of coagulation dysfunction after liver xenotransplantation, the level of TF activation in the primary livers is significantly higher than that in the transplant livers. The TF activation in the primary livers is the main cause of coagulation dysfunction after liver xenotransplantation.