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目的 :观察 NF- κB“decoy”ODNs在阳离子脂质体 lipofectin介导下转染小鼠巨噬细胞 J774 .1的优化参数以及在细胞内的分布。 方法 :改变 ODN与 lipofectin比率、转染时间 ;用流式细胞仪检测细胞内的相对荧光强度和摄取率评价转染效率 ;荧光显微镜观察细胞内荧光分布 ;测定细胞上清乳酸脱氢酶 (L DH)观察细胞损伤情况。 结果 :lipofectin介导转染显著提高 J774 .1细胞对 NF- κB“decoy”ODNs的摄取 ;在 2 4孔培养板中 ,NF- κB“decoy”ODNs与 lipofectin比率 (W/ W)为 1∶ 5、转染 6 h时 ,转染效率最佳而细胞毒性相对较低 ;lipofectin介导转染 6 h后 ,细胞内荧光物质主要聚集于细胞核和部分细胞质中 ,而直接转染时荧光物质多分布于细胞质。结论 :lipofectin可增加 NF-κB“decoy”ODNs的细胞摄入并改变其细胞内分布 ;NF-κB“decoy”ODNs与 lipofectin比率 (W/ W)为 1∶ 5、转染 6 h时可获得最佳转染效率。
Objective: To observe the optimal parameters of J774.1 transfected mouse macrophage NF-κB “decoy” ODNs mediated by cationic liposomes lipofectin and their distribution in intracellular. Methods: The ratio of ODN to lipofectin was changed, and the transfection time was changed. The relative fluorescence intensity and uptake rate in cells were evaluated by flow cytometry to evaluate the transfection efficiency. Fluorescence microscopy was used to observe intracellular fluorescence distribution. Lactate dehydrogenase (L DH) observed cell damage. Results: The uptake of NF-κB “decoy” ODNs was significantly increased by lipofectin-mediated transfection. The ratio of ODNs to lipofectin (W / W) in NF-κB “decoy” 5 transfection at 6 h, transfection efficiency and cytotoxicity is relatively low; 6 hours after lipofectin-mediated transfection, the intracellular fluorescent substances mainly in the nucleus and part of the cytoplasm, and direct transfection of fluorescent substances Distributed in the cytoplasm. Conclusion: lipofectin can increase the cellular uptake of NF-κB “decoy” ODNs and change their intracellular distribution. The ratio of NF-κB “decoy” ODNs to lipofectin is 1: 5, Optimal transfection efficiency.