中文摘要：

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（RN～）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α1，3一半乳糖残基（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Ribo—Juice在12nmol／L：6μl时，干扰效果最佳（67．3％～89．5％），整体量效优于DOSPA及Lipo—fectamine 2000转染体系。靶细胞转染SiRNA-1后48h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6～12h对靶细胞生长稍有影响，24h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。

英文摘要：

Objective To develop an efficient and stable small interfering RNA （SiRNA） transfection protocol and establish an optimized SiRNA delivery system. Methods SiRNAs were biosynthesized in vitro targeting porcine el, 3 galactosyltransferase （αl, 3-GT） gene. After delivering these SiRNAs into pig endothelial cell line PED by three distinct liposome transfection system, DOS- PA, RiboJuice and Lipofectamine 2000, the effect of knock-down Galal-3Galβ1-4GIcNAc-R （α-Gal） expression and viability of PED by RNA interference （RNAi） targeting αl,3-GT were assessed by flow cytometry, trypan blue exclusion and lactate dehydrogenase release assays. Results Specific RNAi phenomenon was observed in PED and displayed in a dose-dependent manner. The ratio of Ribo- Juice transfection reagent and SiRNA-1 at 12 nmol：6μ1 was the most efficient compared to DOSPA and Lipofectamine 2000 transfection systems, reaching 67. 3% - 89. 5% inhibition of a-Gal expres- siorsion. The relative expression level of α-Gal was 10. 5% on PED 48 h post-transfection of SiRNA-1. SiRNA-2, however, did not have any significant effect on the trGal expressiorsion Although liposome tranfection system had a minor cytotoxic effect on the growth of target cells 6 h to 12 h after transfection, PED viability was gradually recovered after 24 h. Conclusions RNAi phenomenon was observed in PED. RiboJuice transfection system was a high efficient and low toxic tool to deliver SiRNA into target cells, which made a foundation for the application of RNAi in suppressing xenograft rejectiortion.