Cell Transplantation and Gene Therapy Institute, The Third Xiang Ya Hospital of Central South University, Changsha, Hunan, China.
Cell Transplant. 2012;21(1):61-71. doi: 10.3727/096368911X580563. Epub 2011 Jun 7.
Tissue factor (TF) expression on islets has been shown to trigger instant blood-mediated inflammatory reaction (IBMIR), leading to rapid islet loss in portal vein islet transplantation. This study investigated whether antisense RNA-mediated TF gene knockdown in islets could suppress IBMIR as a strategy to overcome IBMIR. Neonatal porcine islet cell clusters (NICCs) were transfected with or without TF-specific antisense RNA or a nonspecific RNA by a lipid-based method. Expression of both TF gene and protein in NICCs was analyzed after transfection by real-time PCR, Western blot, and FACS, respectively. The impact of antisense RNA transfection on NICC viability and in vitro function was examined by FACS and insulin release test, respectively. The effect of TF knockdown in NICCs on IBMIR was assessed with an in vitro tubing loop assay using human blood. A significant reduction in TF gene and protein expression was achieved in TF antisense RNA but not control RNA transfected NICCs, which did not affect NICCs' viability or their insulin secreting capacity. Incubation of TF antisense RNA transfected with human blood resulted in a considerable reduction in blood clot formation, platelet consumption, and complement and coagulation activation compared to that observed in the loops containing human blood and untreated or control RNA transfected NICCs. Consistent with these findings, infiltrating neutrophils in the blood clots with entrapped TF antisense RNA transfected NICCs was also reduced substantially compared to that seen in the clots containing untreated or control RNA transfected NICCs. This study presents a nontoxic TF antisense RNA-mediated TF knockdown in porcine islets that leads to an effective suppression of IBMIR, suggesting a potentially new strategy to improve islet transplantation outcomes.
组织因子 (TF) 在胰岛细胞上的表达已被证实可触发即时的血液介导的炎症反应 (IBMIR)，导致门静脉胰岛移植中的胰岛迅速丢失。本研究探讨了在胰岛中通过反义 RNA 介导的 TF 基因敲低是否可以抑制 IBMIR，作为克服 IBMIR 的一种策略。通过脂质方法将新生猪胰岛细胞簇 (NICCs) 与 TF 特异性反义 RNA 或非特异性 RNA 转染。转染后，通过实时 PCR、Western blot 和 FACS 分别分析 NICCs 中 TF 基因和蛋白的表达。通过 FACS 和胰岛素释放试验分别检查反义 RNA 转染对 NICCs 活力和体外功能的影响。通过使用人血的体外管环实验评估 NICCs 中 TF 敲低对 IBMIR 的影响。在 TF 反义 RNA 转染的 NICCs 中，TF 基因和蛋白的表达显著降低，但在对照 RNA 转染的 NICCs 中没有降低，这并不影响 NICCs 的活力或其胰岛素分泌能力。与未处理或对照 RNA 转染的 NICCs 相比，与 TF 反义 RNA 孵育的人血导致血液凝块形成、血小板消耗以及补体和凝血激活显著减少。与这些发现一致的是，与含有未处理或对照 RNA 转染的 NICCs 的凝块相比，TF 反义 RNA 转染的 NICCs 中浸润的中性粒细胞也显著减少。这项研究提出了一种非毒性的 TF 反义 RNA 介导的 TF 敲低方法，可有效抑制 IBMIR，为改善胰岛移植结果提供了一种新策略。
