Zhu Y G, Zhuo G S, Chen Z Z, Chen X C
Fujian Institute of Hematology, Union Hospital, Fujian Medical University, Fuzhou 350001, China.
Acta Pharmacol Sin. 2001 Nov;22(11):1007-12.
To explore the effect of cationic lipid 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER) on cellular uptake and activity of bcl-2 antisense oligodeoxynucleotide G3139 in HL-60 cells.
The cell-associated mean fluorescence intensity and the percentage of cells with positive staining for Bcl-2 were measured by flow cytometry. The subcellular distribution of fluorescein isothiocyanate (FITC)-labeled G3139 was observed by fluorescence microscope and the bcl-2 mRNA level was detected by reverse transcription polymerase chain reaction (RT-PCR).
(1) DOSPER increased cellular uptake of G3139 into HL-60 cells greatly. When DOSPER/G3139 (microg : microg) was 2 : 1, the uptake of G3139 reached top after treatment for 2 h and increased about 20 times compared with application of G3139 alone. In the presence of DOSPER, G3139 was localized in nucleus and cytosol with a bright spotted fluorescence staining. However, G3139 was localized in cytoplasm with faint fluorescence in the absence of DOSPER. (2) Cell-associated G3139 could be effluxed out of cells. After treated with G3139 in the presence of DOSPER for 4 h, the cell-associated G3139 could be fitted by C(t)=68.2e(-0.60) t+31.8e(-0.02) t (% of initial value), with a half-life of approximately 1.1 h. In the absence of DOSPER, the cell-associated G3139 could be fitted by C(t)=64.8e(-2.27) t+35.2e(-0.04) t, with a half-life of about 18 min. (3) In the presence of DOSPER, G3139 1.0 micromol/L specially reduced bcl-2 mRNA level, and Bcl-2 protein decreased from 97 % +/- 4 % to 70.6 % +/- 2.1 %.
DOSPER enhanced the activity of G3139 and it might be attributed to increase of the cellular uptake and change of the subcellular distribution of G3139.
探讨阳离子脂质1,3 - 二油酰氧基 - 2 -（6 - 羧基 - 精胺基） - 丙酰胺（DOSPER）对HL - 60细胞摄取bcl - 2反义寡脱氧核苷酸G3139及细胞内活性的影响。
采用流式细胞术检测细胞相关平均荧光强度及Bcl - 2阳性染色细胞百分比。用荧光显微镜观察异硫氰酸荧光素（FITC）标记的G3139的亚细胞分布，并用逆转录聚合酶链反应（RT - PCR）检测bcl - 2 mRNA水平。
（1）DOSPER显著增加HL - 60细胞对G3139的摄取。当DOSPER/G3139（微克∶微克）为2∶1时，处理2小时后G3139的摄取量达到峰值，与单独应用G3139相比增加约20倍。在有DOSPER存在时，G3139在细胞核和细胞质中呈明亮的点状荧光染色定位。然而，在没有DOSPER时，G3139在细胞质中呈微弱荧光定位。（2）细胞相关的G3139可从细胞中流出。在有DOSPER存在的情况下用G3139处理4小时后，细胞相关的G3139可用C(t)=68.2e(-0.60)t + 31.8e(-0.02)t（初始值的百分比）拟合，半衰期约为1.1小时。在没有DOSPER时，细胞相关的G3139可用C(t)=64.8e(-2.27)t + 35.2e(-0.04)t拟合，半衰期约为18分钟。（3）在有DOSPER存在时，1.0微摩尔/升的G3139特异性降低bcl - 2 mRNA水平，Bcl - 2蛋白从97%±4%降至70.6%±2.1%。
DOSPER增强了G3139的活性，这可能归因于细胞摄取增加及G3139亚细胞分布的改变。
