Prime Tech Ltd, Tsuchiura, Japan.
Xenotransplantation. 2012 Mar-Apr;19(2):82-91. doi: 10.1111/j.1399-3089.2012.00696.x.
For long-term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti-inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK-puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM-PAEC. Although no bleeding tendency was observed in hTM-cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen-antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired.
为了实现长期异种移植物的存活，凝血控制是一个关键问题。我们将注意力转向人血栓调节蛋白（hTM），因为它有望在凝血控制和细胞保护方面发挥以下有益作用：（i）解决蛋白 C 激活中的分子不相容性问题；（ii）仅在形成凝血酶时发挥生理调节剂的作用；（iii）抑制直接凝血酶原酶活性；（iv）具有抗炎作用。使用 pCAGGS 表达载体和 pPGK-puro 载体将 hTM 基因转染到猪（长白/约克夏）成纤维细胞中。经过嘌呤霉素选择后，仅通过细胞分选收集表达高水平 hTM 的成纤维细胞，然后应用于核转移。电激活和随后的培养后，将总共 1547 个分裂胚胎转移到 7 头代孕母猪。两只表达 hTM 的健康克隆猪出生，成功生长至成熟并产生正常后代。F1 代猪的器官免疫组织化学染色显示 hTM 在血管内皮细胞和实质细胞中表达。在肾脏和肝脏的内皮细胞中观察到高表达。从克隆猪的主动脉内皮细胞中发现 hTM 的表达水平与人类脐静脉内皮细胞（HUVEC）相似，并且可以将蛋白 C 转化为活化蛋白 C。人内皮细胞蛋白 C 受体（hEPCR）的阻断显著减少了 HUVEC 中的 APC 产生，但在 hTM-PAEC 中没有。尽管在 hTM 克隆猪中未观察到出血倾向，但活化部分凝血活酶时间（APTT）略有延长，并且在猪血浆中检测到可溶性 hTM。hTM 在血小板和单核细胞中表达，但不在 RBC 中表达。生产出了在血管内皮细胞中表达 hTM 的水平与 HUVEC 相当的克隆猪。由于完全抑制移植物中的抗原-抗体反应对于准确评估与凝血控制相关的转基因至关重要，因此希望生产基于半乳糖基转移酶敲除的表达 hTM 和补体调节蛋白的转基因猪。
