Bcl-2反义寡核苷酸对多药抗性HL-60/HHT细胞耐药性逆转的研究

Reversion of drug resistance in multidrug resistant cell line HL-60/HHT by Bcl-2 antisense oligonucleotides

目的 探讨bcl 2基因在急性白血病细胞凋亡及耐药性中的作用及Bcl 2反义寡核苷酸(ASON)逆转耐药性的效果。方法 细胞耐药性测定采用MTT法 ,Bcl 2和Bax蛋白含量测定采用单克隆抗体标记技术在流式细胞仪上测定 ,细胞凋亡率采用流式细胞仪测定。结果 (1)Bcl 2水平在HL 6 0 HHT细胞系明显高于HL 6 0细胞系 (P <0 0 1) ;(2 )Bcl 2ASON可逆转HL 6 0 HHT的耐药性 (P<0 0 1) ;(3)HL 6 0 HHT细胞系的凋亡百分率经ASON作用明显升高 (P <0 0 5 ) ,经ASON +HHT作用后升高更加显著 (P <0 0 1) ;(4)Bcl 2ASON可明显降低Bcl 2表达水平 (P <0 0 1)。结论 在HL 6 0 HHT细胞系中 ,Bcl 2的高度表达是造成其耐药的重要原因 ;反义技术可以通过下调Bcl 2 Bax比值 ,部分逆转它的耐药的发生。通过进一步的深入研究 ,反义技术将有可能应用于难治及耐药的急性白血病。

Objective Multidrug resistance is one of the important causes of refractoriness or relapse in children with acute leukemia Recently, it was proved that the increased level of Bcl 2 is an important reason leading to drug resistance. To probe into the role of the Bcl 2 gene in the antiapoptosis and drug resistance of acute leukemia and the effect of Bcl 2 antisense oligonucleotide (ASON) on reversing multidrug resistance Methods The influence of Bcl 2 ASON on the chemosensitivity was determined by MTT assay The Bcl 2 and Bax protein levels in HL 60 and HL 60/HHT were detected by McAb direct label method with FACScan flow cytometer The apoptosis of HL 60/HHT cell lines was measured by FACS The experiment was designed to have following groups, HHT group, ASON plus HHT group, nonsense oligonucleotides plus HHT group. Results (1) The levels of Bcl 2 protein in HL 60/HHT(92±6) was significantly higher than that in HL 60(61±4) ( P <0 01) (2) The Bcl 2 ASON could significantly reverse the chemosensitivity of HL 60/HHT( P <0 01) The IC 50 of HHT decreased from 89 ng/ml to 51 ng/ml( P <0 01); The Bcl 2 ASON could increase the apoptosis of HL 60/HHT (3)The Bcl 2 ASON could increase the apoptosis of HL60/HHT The percentage of apoptosis of HL 60/HHT was higher when exposed in ASON (19 4±1 9)% than that exposed in HHT [(14±1 9)%, P <0 05], and much higher when exposed in HHT+ASON[ (46±10)%, P <0 01] (4)The Bcl 2 level could be reduced by ASON ( P <0 01) The Bcl 2 levels were 92±6 in control group, 84±6 in HHT group, 71±6 in ASON group ( P <0 05), and 44±5 in ASON plus HHT group ( P <0 01). Conclusion High level of Bcl 2 protein in HL 60/HHT cell line may be one of the important causes of the drug resistance ASON could reduce the Bcl 2 level and partialy reverse the drug resistance. The results suggested that antisense technique would be applied hopefully to treat refractory or drug resistant acute leukemia after further studies