VEGF反义核酸对 HL-60细胞作用和机制研究

本研究探讨血管内皮生长因子(VEGF)反义核酸对HL-60细胞生长的量效和时效关系，并研究其作用机制以揭示VEGF基因新的功能。用含20个脱氧核糖核酸的反义核酸A7(用计算机模拟设计和实验筛选出的最优序列)全硫代修饰，以脂质体介导进入细胞，培养72小时后用MTT法计算细胞生长抑制率；采用台盼蓝拒染法每24小时观察细胞存活情况并计数；用Giemsa染色观测细胞形态学改变；用流式细胞仪检测细胞凋亡百分数；用VEGF ELISA试剂盒测定蛋白的水平。结果显示，反义药物对HL-60细胞生长有明显抑制作用，呈剂量依赖关系，并且下调VEGF蛋白的表达；反义药物对HL-60细胞的凋亡无明显影响。结论：VEGF反义药物抑制HL-60细胞生长的机制是抑制细胞增殖，而不是促进细胞凋亡，内源性VEGF蛋白具有促进HL-60细胞增殖的功能。

Effect and Mechanism of VEGF Antisense Phosphorothioate Deoxynucleotides on HL-60 Leukemic Cells

To explore dose-effect or time-effect of vascular endothel ial growth factor (VEGF) antisense phosphoro- thioate oligodeoxynucleotides (AS PS-ODN) on growth of HL-60 cells, and to study the effect mechan ism so as to find new role of VEGF, A7, which was the most effective one of AS P S-ODN selected with computer-aided design and experimental assay, contains 20-DNA m odified with phosphorothioate and was tranfered into cells mediated with lipofec tin. After culture for 72 hours, inhibitive rate of cell growth was detected with MTT methods, v iable cells were counted with trypan blue exclusion each 24 hour, cell configura tion and apoptosis were observed with Geimsa staining and flow cytometry respect ively, level of VEGF protein was detected with VEGF EL ISA kit. The results showed that A7 is able to inhibit cell growth of HL-60 in dose-depending manner of AS PS-ODN, to down-regulate VEGF protein express ion sig nificantly, and not to induce apoptosis of HL-60 cells. It is concluded that there is possibility that the inhibition effect of VEGF AS PS-ODN on HL-60 cell growth is t o restrain cell proliferation without inducing apoptosis of HL-60 cell , which would interpret that endogenous VEGF proteins have a capacity of promoting p roliferation of HL-60 cell.