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Establishment of an optimized small interfering RNA transfection system for porcine endothelial cells
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF ORGAN TRANSPLANTATION
- Issue:
- 2
- DOI:
- 10.3760/cma.j.issn.0254-1785.2008.02.001
- Key Word:
- RNA干扰;半乳糖苷酶类;内皮细胞
Abstract： 目的 探索建立高效、稳定的小分子干扰RNA(SiRNA)转染方案并建立优化的猪血管内皮细胞RNA干扰(RNAi)转染体系.方法 针对猪α1,3-半乳糖苷转移酶(α1,3-GT)基因合成数对SiRNA分子,运用不同转染体系(DOSPA、RiboJuice及Lipofectamine 2000)转染永生化猪血管内皮细胞系(PED).分别应用流式细胞术、台盼蓝拒染试验及乳酸脱氢酶释放试验评价干扰α1,3-GT作用下PED的α1,3-半乳糖残基(α-Gal)表达情况以及SiRNA-脂质体转染对PED生长的影响.结果 PED能有效发生特异性RNA干扰现象,且呈明显的剂量依赖性特征.SiRNA-1与脂质体RiboJuice在12 nmol/L∶6 μl时,干扰效果最佳(67.3%～89.5%),整体量效优于DOSPA及Lipofectamine 2000转染体系.靶细胞转染SiRNA-1后48 h时的α-Gal相对表达水平为10.5%,而SiRNA-2转染组则未见明显干扰现象.脂质体转染体系在转染后6～12 h对靶细胞生长稍有影响,24 h后细胞活性逐渐恢复.结论 猪血管内皮细胞存在RNA干扰机制,RiboJuice转染体系是一种高效、低毒的SiRNA转染方式,为研究RNAi在抑制异种排斥反应中的应用奠定了基础.
- [1]Michelle Holko,Michael J. de Veer,Robert H. Silverman,etc.Activation of the interferon system by short-interfering RNAs[J].2003,5(9).
- [2]Ulrike S.Eggert,Timothy J.Mitchison,Christine M.Field.Small molecules in an RNAi world[J].2006,2(2).
- [3]Wall NR,Shi Y.Small RNA: can RNA interference be exploited for therapy?[J].2003,362(9393).
- [4]Iwona Wrobel,David Collins.Fusion of cationic liposomes with mammalian cells occurs after endocytosis[J].1995,1235(2).
- [5]Kuhnel F,Trautwein C,Gosling T,etc.Caspase 8 small interfering RNA prevents acute liver failure in mice.[J].2003,100(13).
- [6]Z E, Holzknecht,J L, Platt.Identification of porcine endothelial cell membrane antigens recognized by human xenoreactive natural antibodies.[J].1995,154(9).