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目的 了解ＷＴ１ 反义寡核苷酸对白血病细胞增殖和凋亡的影响。方法 应用ＷＴ１ 基因的反义寡核苷酸（ＷＴ１ ＡＳＯ）处理Ｋ５６２、Ｕ９３７、ＨＬ６０ 细胞系，白血病患者以及正常人骨髓细胞，然后以台盼蓝拒染法、细胞集落法及流式细胞仪检测法确定白血病细胞的增殖和凋亡。结果 ＷＴ１ ＡＳＯ 能够抑制ＷＴ１ 表达阳性的Ｋ５６２ 细胞生长，对ＷＴ１ 表达阴性的Ｕ９３７ 细胞则无抑制作用；ＷＴ１ ＡＳＯ对８ 例急性髓系白血病患者中４ 例的白血病细胞集落（ＣＦＵＬ）有显著抑制作用，对８ 例正常对照骨髓细胞的ＣＦＵＧＭ 则无抑制作用；而ＷＴ１ 有义寡核苷酸（ＳＯ） 对白血病细胞系、白血病患者的ＣＦＵＬ以及正常对照骨髓ＣＦＵＧＭ 均无抑制作用。ＷＴ１ ＡＳＯ可以引起Ｋ５６２ 细胞的凋亡，随着作用时间的延长凋亡数增加，结合小剂量Ｖｐ１６（１０ μｇ／ｍｌ） 后细胞凋亡率明显提高；ＨＬ６０ 细胞经ＷＴ１ ＡＳＯ处理２４ 小时及６０ 小时均无明显凋亡，接近有义链组及对照组，但ＷＴ１ ＡＳＯ与Ｖｐ１６（１０ μｇ／ｍｌ）联合处理６０ 小时后细胞凋亡增加，大于单独应用Ｖｐ１６ 及Ｖｐ１６＋ ＳＯ引起的凋亡。结论 ＷＴ１ 反义寡核苷酸对白血病细胞有特异的抑制?
Objective To investigate the effect of WT1 antisense oligonucleotides on proliferation and apoptosis of leukemia cells. Methods K562, U937, and HL-60 cell lines were treated with WT1 gene antisense oligonucleotide (WT1 ASO), leukemia patients and normal human bone marrow cells, and then trypan blue exclusion, cell colony formation, and flow cytometry were used. The assay was used to determine the proliferation and apoptosis of leukemic cells. Results WT1 ASO could inhibit the growth of K562 cells positive for WT1 expression, but had no inhibitory effect on U937 cells negative for WT1 expression; WT1 ASO significantly inhibited leukemia cell colonies (CFUL) in 4 of 8 acute myeloid leukemia patients. The effect of CFUGM on bone marrow cells in 8 normal controls was not inhibited; WT1 sense oligonucleotides (SO) did not inhibit leukemia cell lines, CFUL in leukemia patients, and CFUGM in normal control bone marrow. effect. Apoptosis of K562 cells was induced by WT1 ASO, and the number of apoptotic cells increased with the prolonged action time. Apoptosis rate was significantly increased when combined with a small dose of Vp16 (10 μg/ml); HL60 cells were treated with WT1 ASO for 24 hours and There was no obvious apoptosis at 60 hours, which was close to the sense chain group and the control group. However, the apoptosis of WT1 ASO and Vp16 (10 μg/ml) combined treatment for 60 hours was greater than that of Vp16 and Vp16+ SO alone. Conclusion WT1 antisense oligonucleotide has specific inhibition on leukemia cells.