摘要
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 search for the optimizing parameters and distribution pattern of NF κB decoy oligodeoxynucleotides in transfecting J774.1 cells mediated by lipofectin. Methods: With the change of ODNs/lipofectin ratio and transfection time, the uptake rate and mean fluorescence intensity of NF κB decoy oligodeoxynucleotides in J774.1 cells were measured by flow cytometry to evaluate transfection efficiencies. Intracellular distribution of NF κB decoy oligodeoxynucleotides was determined with fluorescence microscopy. The lactate dehydrogenase (LDH) activity in the supernatant was assayed to assess the cytotoxicity. Results: The uptake of NF κB decoy oligodeoxynucleotides into J774.1 cells was significantly improved by lipofectin. In 24 well culture plate, 1 μg ODNs with 5μg lipofectin ( W/W =1∶5) resulted in the highest transfection efficiency and lower cytotoxicity. The NF κB decoy oligodeoxynucleotides localized in the nucleus as well as in the cytoplasm following an incubation of 6 h with lipofectin. While NF κB decoy oligodeoxynucleotides had faint fluorescences in cytoplasm in the absence of lipofectin. Conclusion: lipofectin can enhance the cellular uptake of NF κB decoy oligodeoxynucleotides in J774.1 cells and alter intracellular distribution of NF κB decoy oligodeoxynucleotides. Efficiency of transfection is the highest when the ratio of ODNs/lipofectin is 1∶5 for incubation of 6 h.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2003年第8期856-858,F004,共4页
Academic Journal of Second Military Medical University
基金
上海市教委曙光计划
