CAJ | 학술논문

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-60/HHT细胞系明显高于HL-60细胞系（P＜0.01）；（2）Bcl-2ASON可逆转HL-60/HHT的耐药性（P＜0.01）；（3）HL-60/HHT细胞系的凋亡百分率经ASON作用明显升高（P＜0.05），经ASON+HHT作用后升高更加显著（P＜0.01）；（4）Bcl-2ASON可明显降低Bcl-2表达水平（P＜0.01）。结论在HL-60/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。
목적탐토bcl-2기인재급성백혈병세포조망급내약성중적작용급Bcl-2반의과핵감산（ASON）역전내약성적효과。방법세포내약성측정채용MTT법，Bcl-2화Bax단백함량측정채용단극륭항체표기기술재류식세포의상측정，세포조망솔채용류식세포의측정。결과（1）Bcl-2수평재HL-60/HHT세포계명현고우HL-60세포계（P＜0.01）；（2）Bcl-2ASON가역전HL-60/HHT적내약성（P＜0.01）；（3）HL-60/HHT세포계적조망백분솔경ASON작용명현승고（P＜0.05），경ASON+HHT작용후승고경가현저（P＜0.01）；（4）Bcl-2ASON가명현강저Bcl-2표체수평（P＜0.01）。결론재HL-60/HHT세포계중，Bcl-2적고도표체시조성기내약적중요원인；반의기술가이통과하조Bcl-2/Bax비치，부분역전타적내약적발생。통과진일보적심입연구，반의기술장유가능응용우난치급내약적급성백혈병。
Objective Multidrug resistance is one of the important causes ofrefractoriness 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.