siRNA阻断猪内皮细胞组织因子表达对异种移植中抗凝作用影响的研究

Effect on anticoagulation by siRNA-mediated tissue factor knockdown in pig aortic endothelial cells in vitro

目的探讨在异种移植中,siRNA阻断猪内皮细胞组织因子表达后对细胞抗凝作用的影响。方法 siRNA转染猪内皮细胞后,real-time PCR及流式细胞仪分别检测组织因子的基因及蛋白表达水平;并将转染与未转染siRNA的内皮细胞分别与人血液共孵育60 min,检测凝血块生成及血液中血细胞数量。结果 re-al-time PCR及流式细胞仪检测siRNA转染后,猪内皮细胞组织因子的基因及蛋白表达水平明显降低,与人血液共同孵育60 min后,siRNA转染组和未转染组的凝血块生成和血液中血细胞计数没有明显的差异。结论在异种移植中,猪内皮细胞组织因子表达的降低没有明显的增强内皮细胞抗凝作用。

【Objective】 To study the effect on anticoagulation of pig aortic endothelial cells（SVAP） by siRNA-mediated tissue factor（TF） knockdown in vitro.【Methods】 SVAP were transfected with siRNA specific for TF or a nonspecific control siRNA using lipofectamine 2,000.Transfected SVAP were then analyzed for TF gene and protein expression by real-time PCR and FACS respectively.The effect of TF knockdown in SVAP on anti-coagulation was evaluated by incubation with human blood for 60 min.Clots were collected and measured.Platelets,leukocytes and neutrophils were counted by using Beckman Coulter ACT.【Results】 TF siRNA transfection of SVAP resulted in substantially reduced expression of both TF gene and protein expression.Nonspecific siRNA had no effect.When TF siRNA transfected SVAP were incubated with human blood there was no obvious reduced clot weight and reduced consumption of platelets,leukocytes and neutrophils when compared to that detected in the well with non-transfected NICC.【Conclusion】 The expression of TF on endothelial cells is involved in coagulation reaction but not the major coagulation factor in xenotransplantation.