siRNA Mediated Knockdown of Tissue Factor Expression in Pigs for Xenotransplantation
siRNA Mediated Knockdown of Tissue Factor Expression in Pigs for Xenotransplantation
复制标题
DOI：
10.1111/ajt.13120
复制
发表时间：
2015-05-01
影响因子：
8.8
通讯作者：
Niemann, H.
中科院分区：
文献类型：
--
作者：
Ahrens, H. E.;Petersen, B.;Niemann, H.
急性血管排斥反应（AVR），特别是微血管血栓形成，是成功的猪到灵长类动物异种移植的重要障碍。在这里，我们报告的小干扰（si）RNA介导的基因沉默诱导的组织因子（TF）水平下降的猪的一代。用TF靶向小发夹（sh）RNA转染猪成纤维细胞，并用于体细胞核移植。对子代进行siRNA、TF mRNA和TF蛋白水平分析。通过全血凝血试验和流动室试验研究TF下调的功能。TF siRNA在所有12只活产仔猪中表达。与野生型（WT）相比，TF敲低（TFkd）成纤维细胞中TF mRNA表达降低94.14.7%。在TFkd猪中，用50 ng/mL TNF-α刺激的PAEC中的TF蛋白表达显著较低（平均荧光强度TFkd：7136 +/- 136 vs. WT：13038 +/- 1672）。TF下调显著增加凝血时间（TFkd：73.3 +/- 8.8min，WT：45.8 +/- 7.7min，p
Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.14.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50ng/mL TNF- was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 +/- 136 vs. WT: 13038 +/- 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 +/- 8.8min, WT: 45.8 +/- 7.7min, p