血管内皮生长因子反义寡核苷酸对白血病细胞系HL-60细胞作用的研究

Effect of Vascular Endothelial Growth Factor Antisense Oligonucleotide on Human Leukemic Cell Line HL-60

本研究探讨VEGF ASODN抑制白血病细胞内源性VEGF mRNA的表达作用及其与白血病细胞凋亡的关系。用阳离子聚合物介导硫代修饰VEGF ODN转染体外培养白血病细胞系HL-60细胞后,采用MTT法检测细胞增殖抑制率,RT-PCR检测VEGF mRNA表达,以细胞形态学,凝胶电泳,流式细胞术观察细胞凋亡。结果显示:反义组与错义组、空白对照组相比,在细胞增殖抑制率和VEGF mRNA的表达水平方面均具有显著性差异(p<0.05);错义组与空白对照组相比无统计学差异(p>0.05)。反义组可见细胞皱缩,颗粒增多,核固缩并出现细胞碎片,而错义组、空白对照组细胞轮廓清楚,胞体透亮,生长旺盛;反义组有凋亡梯形带,错义组和空白对照组仅见1条DNA条带;反义组细胞凋亡率达19.46%,与错义组、空白对照组相比具有显著性差异(p<0.05);错义组与空白对照组相比,无统计学差异(p>0.05)。VEGF ASODN联合VP16的细胞凋亡率与等同条件单用VP16组相比有统计学差异(p<0.05);且凋亡率具有时间和剂量依赖关系。结论:VEGF ASODN能抑制白血病细胞内源性VEGF mRNA的表达,诱导白血病细胞的凋亡,抑制增殖,且增强VP16对白血病细胞诱导凋亡作用,两者联合效应具有相加作用。

This study was aimed to investigate expression of vascular endothelial growth factor mRNA （VEGF mRNA） and its relationship with leukemic cell apoptosis after VEGF antisense oligonucleotide （VEGF ASODN ） transferred into HL-60 cells. The phosphorothiate VEGF ODN was transferred into HL-60 cells in vitro by using cation poly mediated method, the inhibitory rate of cell proliferation was assayed by MTT, expression of VEGF mRNA was measured by RT-PCR, cell apoptosis was detected by cell morphology observation, DNA agarose gel electrophoresis and flow cytometer （FCM）. The results showed that difference of the inhibitory rate of cell proliferation and the relative expression of VEGF mRNA between ASODN group and MSODN or control groups under the same condition（p 〈 0.05 ） was statistic significant, but no significant difference （p 〉0.05 ） was found between MSODN and control. The number of clusters of cells in ASODN group decreased; the morphology features of apoptotic cells involved cell shrinking, more granulation in cytoplasm, nuclear contracting and many fragments of cells. In MSODN and control groups, however, cells were plump and clear, and grow healthly. The result of electrophoresis revealed DNA ladder in ASODN group, while only one band of DNA in control groups. The rate of cell apoptosis was 19. 46% in ASODN group with a significant difference as compared with MSODN groups and control （p 〈 0.05 ）. The rate of HL-60 cell apoptosis in combination of VEGF ASODN with VP16 was significantly higher than that in VP16 alone （p 〈 0.05 ） and showed time-and dose- dependence. It is concluded that VEGF ASODN can down-regulate expression of VEGF mRNA of HL-60 cells, induces the apoptosis , inhibits the proliferation of HL-60 cells and enhances VP16-induced apoptosis in HL-60 cells, the VEGF ASODN in combination with VP16 shows additive effect.