bcr-ahl融合基因反义寡核苷酸对K562细胞生长的影响

The effect of antisense bcr - abl oligonucleotides on the growth of K562 cells

目的：研究 ｂｃｒ－ａｂｌ融合基因硫代磷酸反义寡核苷酸（Ａｓｐｏ）对 Ｋ５６２细胞生长特性的影响，探讨其在慢性粒细胞白血病（ＣＭＬ）基因治疗中的意义。方法：倒置显微镜下细胞活体观察；Ａｓｐｏ与细胞共同培养２４ｈ、４８ｈ、７２ｈ、９６ｈ及 １２０ｈ，用０．４％台盼蓝染色计各处理组死活细胞数，甲基纤维素半固体培养法观察其克隆形成率的变化，流式细胞仪测定各处理组细胞Ｐ２１０蛋白表达变化。结果：Ａｓｐｏ处理后细胞仍呈克隆状生长，当 Ａｓｐｏ浓度达５μｍｏｌ／Ｌ时，即可产生增殖抑制作用，呈一定的量效关系，其最大抑制效应时间为作用１２０ｈ，当Ａｓｐｏ达１０μｍｏｌ／Ｌ时，在一定细胞浓度范围内（１×１０４／ｍＬ－ ５× １０５／ｍＬ），均有显著抑制作用，当Ａｓｐｏ浓度达５μｍｏｌ／Ｌ以上时，Ｋ５６２细胞 Ｐ２１０蛋白表达明显下降甚至不表达。 ｂ２ａ２型 Ａｓｐｏ对表达 ｂ３ａ２型 ｍＲＮＡ的 Ｋ５６２细胞也表现出交叉抑制作用。结论：ｂｃｒ－ａｂｌ融合基因反义寡核苷酸对Ｋ５６２细胞具有特异性增殖抑制作用，在慢性粒细胞白血病基因治疗中值得深人研究。

AIM:To study the effect of bcr - abl gene antisense phosphorothioate oligonucleotides(Aspo) on K562 cell line and explore its significance in chronic myelogencous leukemia (CML) gene therapy. METHODS: Cells were exposed to oligomers, observed by inverted microscope. Cells inhibitory rate were determined by 0.4% trypan blue exclusion. CFU - K562 were cultured in 0.8% methylcellulose. P210 was measured by flow cytometry. RESULTS:K562 cells were treated with Aspo, they still grew in clone state and show antisense sequence specific and dose dependent. When the concentration of Aspo was more than 5μmol/L, the growth of cells was inhibited and P210 was down regulated or completely suppressed, and the greatest growth inhibition was at 120h. There was significant inhibition of cell proliferation in a rang of cells number from 1 × 10-4/mL to 5 × 10-4/mL after treatment with 10μmol/L Aspo. b2a2 Aspo was also effect on K562 cells which expressing b3a2 mRNA. CONCLUSION: bcr - abl Aspo has a specific growth inhibition effect on K562 cells, and worths further study in CML gene therapy.