论文部分内容阅读
目的探讨髓系白血病-1(Mcl-1)基因在HL60细胞对全反式维甲酸(ATRA)耐药中的作用。方法采用少量、递增、反复ATRA诱导HL-60细胞,建立ATRA耐药细胞系HL60/ATRA;利用鸡尾酒法制备Mcl-1基因的小片段干扰RNA(SmallinterferenceRNA,siRNA),并通过脂质体介导将其导入HL60/ATRA细胞;Westernblot检测Mcl-1蛋白在细胞中的表达;MTT实验、NBT还原反应实验、TUNEL免疫组化染色法分别检测细胞的增殖、分化和凋亡情况。结果HL60/ATRA细胞Mcl1蛋白相对灰度值为0.624±0.127,较野生型HL60细胞(相对灰度值为0.162±0.127)明显高表达,并可耐受100nmol/L的ATRA,而不发生分化、凋亡[凋亡率为(1.0±0.5)%];Mcl1siRNA导入HL60/ATRA细胞后,Mcl1蛋白表达下调(相对灰度值为0.267±0.086),恢复对ATRA的敏感性[凋亡率为(18.5±4.5)%]。结论Mcl1基因可能参与了HL60细胞ATRA耐药的形成;抑制Mcl-1基因可能成为一种新的逆转ATRA耐药的策略。
Objective To investigate the role of myeloid leukemia-1 (Mcl-1) gene in the resistance of all-trans retinoic acid (ATRA) cells to HL60 cells. METHODS: ATRA-induced HL60 / ATRA cells were induced by ATRA in a small amount and with increasing doses. A small interfering RNA (siRNA) of Mcl-1 gene was prepared by a cocktail method. The cells were transfected into HL60 / ATRA cells. The expression of Mcl-1 protein in cells was detected by Western blot. The proliferation, differentiation and apoptosis of M60 cells were detected by MTT assay, NBT reduction assay and TUNEL immunohistochemical assay. Results The relative gray value of Mcl1 protein in HL60 / ATRA cells was 0.624 ± 0.127, which was significantly higher than that in wild-type HL60 cells (relative gray value was 0.162 ± 0.127) and tolerated 100 nmol / L ATRA without differentiation, (1.0 ± 0.5%). The Mcl1 protein expression was down-regulated (relative gray value was 0.267 ± 0.086) after Mcl1 siRNA was transduced into HL60 / ATRA cells, and the sensitivity to ATRA was restored [ 18.5 ± 4.5)%]. Conclusion The Mcl1 gene may be involved in the formation of ATRA resistance in HL60 cells. Inhibition of Mcl-1 gene may be a new strategy to reverse ATRA resistance.