Study on SCARB1 mediated coagulation dysregulation in recipients after liver xenotransplantation
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摘要:
目的 探讨清道夫受体B组1型分子（SCARB1）在异种肝移植手术前、后移植肝组织内的表达变化及其与受体凝血功能调节障碍的关系。 方法 以α-1, 3-半乳糖苷转移酶基因敲除（GTKO）五指山小型猪为供体，藏酋猴为受体，建立异种异位辅助性肝移植模型，并收集移植手术前、后移植肝脏组织标本。以胶原酶消化法提取原代猪肝细胞，免疫磁珠分离人外周血单核细胞，将两者混合培养并加入人血浆，建立异种肝移植术后凝血功能调节障碍的细胞模型。以实时定量聚合酶链反应（RT-PCR）和蛋白印迹法（Western blot）检测并对比组织及细胞标本中SCARB1的信使核糖核酸（mRNA）及蛋白质表达情况。最后在细胞水平以慢病毒载体干预SCARB1的表达，并检测凝血时间以验证其对凝血功能的影响。 结果 与术前组织标本比较，术后移植肝组织内SCARB1的mRNA和蛋白表达均显著下调（均为P＜0.05）。在细胞模型中，与未干预猪肝细胞比较，经人单核细胞共培养后的猪肝细胞内SCARB1的mRNA和蛋白表达也出现显著下调（均为P＜0.05）。与干预组比较，应用慢病毒过表达猪肝细胞内SCARB1的表达后，凝血时间显著延长（均为P＜0.05）。 结论 移植肝组织内SCARB1的下调是介导受体凝血功能调节障碍的重要原因之一，对其干预有助于纠正异种肝移植术后受体凝血功能调节障碍。 Abstract:Objective To investigate the changes in the expression levels of scavenger receptor class B member 1 (SCARB1) in the liver tissues before and after liver xenotransplantation and analyze the relationship between the variations in the SCARB1 expression and coagulation regulating dysfunction in the recipients. Methods The Wuzhishan miniature pig with α-1, 3-galactosyl-transferase gene-knockout (GTKO) was utilized as the donor and Macaca thibetana was chosen as the recipient. Heterotopic auxiliary liver xenotransplantation models were established. The liver tissue specimen was collected before and after liver xenotransplantation. Primary hepatocytes were extracted from the pig using collagenase digestion method. Human peripheral blood mononuclear cells were obtained by immunomagnetic bead sorting. These two types of cells were co-cultured and supplemented with human plasma to establish cell models with coagulation regulating dysfunction following liver xenotransplantation. Quantitative real-time polymerase chain reaction (RT-PCR) and Western blot were performed to quantitatively measure and statistically compared the expression levels of messenger ribonucleic acid (mRNA) and protein of SCARB1 in the tissue and cell samples. At the cellular level, the expression of SCARB1 was interfered by lentiviral vector. The coagulation time was detected to validate the effect upon coagulation function. Results The expression levels of SCARB1 mRNA and protein were significantly down-regulated after liver xenotransplantation (both P < 0.05). In the cell models, the expression levels of SCARB1 mRNA and protein in the porcine hepatocytes co-cultured with human monocytes were significantly down-regulated compared with those in porcine hepatocytes without intervention (both P < 0.05). Compared with the non-intervention group, the coagulation time was significantly prolonged after the expression of SCARB1 was interfered by lentiviral vector (P < 0.05). Conclusions The down-regulated expression of SCARB1 in the liver graft is one of the main causes of mediating coagulation regulating dysfunction. Intervention of SCARB1 expression contributes to resolve the coagulation regulating dysfunction in the recipients after liver xenotransplantation. -
图 1 移植手术前、后移植肝组织内SCARB1蛋白质表达水平的比较
Figure 1. Comparison of SCARB1 protein expression in transplant liver tissue before and after transplantation
图 2 共培养前后肝细胞中SCARB1蛋白表达水平的比较
Figure 2. Comparison of SCARB1 protein expression in hepatocytes before and after co-culture
图 3 各组肝细胞中SCARB1蛋白表达水平的比较
Figure 3. Comparison of SCARB1 protein expression in hepatocytes of each group
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