bcl-2反义寡聚脱氧核苷酸抑制HL-60细胞生长的研究

Antisense Oligodeoxynucleotide to bcl - 2 Gene Inhibits Proliferation of HL - 60 Cells

在体外培养中研究了基因的反义寡聚脱氧核苷酸(antisense oligodeoxynucleo-tide,ASON)对HL-60细胞生长增殖的影响。根据bcl-2基因cDNA的序列分析,以bcl-2mRNA翻译起始区域前15个核苷酸为作用靶序列,人工合成了15聚ASON片段,同时合成15聚无关寡聚核苷酸(ODN)片段,以作序列特异性对照。把上述ASON和无关ODN片段与HL-60细胞共培养,同时设置空白对照组,作用一定时间后分别测定细胞动力学和~3H-TdR掺入率。实验发现:从3.5—30.0μmol/L的ASON对细胞生长有不同程度抑制作用,低于10μmol/L时抑制作用小,大于10.0μmol/L时抑制作用显著,浓度愈大,抑制程度愈大,而无关ODN作用组的细胞生长状态与空白对照组无显著差别,作用48小时后浓度3.5μmol/L抑制率为6％,而20.0μmol/L时上升为40％;作用96小时后3.5μmol/L的ASON抑制率是5.1％,30μmol/L的抑制率高达67％。相同浓度的ASON作用不同时间后,细胞生长率各不相同,当浓度达到14.0μmol/L时,48小时的细胞生长抑制率达22％,72和96小时的抑制率分别为25％和29.3％。HL-60细胞被ASON处理后,培养体系中死细胞数随时间延长而增加,浓度在10—30μmol/L范围内,死细胞率由2％增到50％,但在无关ODN及空白对照组中未观察到这种现象。

Studies have shown that specific antisense oligodeoxynucleotides targeted against oncogenes can inhibit expression of these given cellular genes, as well as the cell growth. bcl - 2 is a novel cellular oncogene, which has been believed to possess the effect of blocking programmed cell death (apoptosis) in lymphoid and hematopoietic tissues. Studies demorstrated that bcl - 2 gene is operative in some kinds of lymphoma and leukemia. HL -60 celi line, human progranulocytic leukemia cells line, con-tains higher Ievel expression of bcl - 2 genes. In order to investigate the effect of bcl - 2 expression de-cline on HL - 60 celis, we utilize artificially synthesized bcl - 2 mRNA specific antisense oligomer (15nt) to coculture with HL - 60 celis, which aimed at observing the status of cellular growth, prolif-eration and death. Using determination of cellular kinetics, typan - blue staining and 3H - TdR in-corporation, we conclude as followed: ①bcl - 2 antisense oligomer notably arrests HL - 60 cell growth and proliferation; ②Such effect shows dose - dependent manner, for example, celi growth is inhibited at rate of 40% at concentration of 20 μmol/L for 48 hours, at 30 μmol/L for 48 hours it reach the rate of 58% ; ③Time- effect relationship can also be observed; ④The rate of 3H- TdR incorporation in HL- 60 cells is decreased when the antisense oligomer challenge celis; ⑤Adding antisense oligomer in HL - 60 cells results in not only the appearing of apoptotic bodies in cells that were observered on elec-ton microscope, but also the electrophoresis of DNA ladder, which is representative of apoptosis. In conclusion, this study suggest bcl - 2 specific antisense oligomer could induce occuring of apoptosis in HL -60 celis, therefore the gene, bcl -2 might be an useful target in gene therapy of leukemia.