脂质体转染bcl-2反义寡核苷酸G3139在HL-60细胞中的动力学模式

Kinetics of cellular uptake and distribution of 5'-FITC labeled bcl-2 antisense oligodeoxynucleotide G 3139 transfected by cationic liposome in HL-60 cells

目的 应用阳离子脂质体介导或直接转染时 ,建立HL 6 0细胞摄入由 5 FITC标记的bcl 2反义寡核苷酸G3139的动力学模式及观察G 3139在细胞内的分布。方法 流式细胞仪测定细胞内的相对平均荧光强度 ,荧光显微镜观察细胞内的荧光分布。结果 ①脂质体介导转染法显著提高HL 6 0细胞对G 3139的摄入 ,当DOSPER/G 3139(μg/μg)为 2 2 / 1时 ,细胞内相对平均荧光强度可达G 3139直接作用时的 40倍 ,而且HL 6 0细胞对G 3139的摄入跟G3139的浓度和作用时间有关。②细胞内的G 3139可向胞外转运 ,在DOSPER介导转染后 ,胞内荧光强度衰减缓慢 ,t1/ 2 约为 4h ,而G 3139直接作用后 ,t1/ 2 约为 0 5h ;③DOSPER介导转染 6h后 ,细胞内荧光物质主要以明亮的点状聚集在细胞核和部分细胞浆的细胞器中 ,而G 3139直接作用时 ,荧光物质则弥漫分布于胞浆中。结论 DOSPER可以增加G 3139的细胞摄入并改变其胞内分布 ,可能是阳离子脂质体增加bcl 2反义寡核苷酸生物活性的重要原因。

AIM To investigate the kinetics of cellular uptake and distribution of 5-fluorescein labled bcl-2 antisense oligodeoxynucleotide G 3139 with or without cationic liposome DOSPER in HL-60 cells. METHODS The mean intracellular fluorescence intensity of G 3139 were measured by flow cytometry and intracellular distribution of G 3139 was evaluated with fluorescence microscopy.RESULTS The uptake of G 3139 into HL-60 cells was greatly improved by DOSPER.When DOSPER/G 3139(μg/μg)was 2 2/1, the uptake of G 3139 in HL-60 cells increased about 40 times compared with application of G 3139 alone.Accumulation of the G 3139 was time and concentration dependent in the absense or presence of DOSPER. The labeled G 3139 were effluxed from HL-60 cells rapidly( t 1/2 =0 5 hour) in the absence of DOSPER,significantly different from the condition with DOSPER ( t 1/2 =4 hours). The G 3139 locatized within the nucleus as well as in the cytoplasm following an incubation of 6 hours with DOSPER.Otherwise,G 3139 localized in cytoplasm with faint fluorescencee in the absence of DOSPER. CONCLUTION DOSPER can enhance the cellular uptake of G 3139 in HL-60 cells and alter the intracellular distribution of G 3139.It is a potential tool to enhance activity of antisense drug.