淀粉样前体蛋白基因在急性髓系白血病的表达及其生物学行为研究

Expression and role of amyloid precursor protein gene in acute myeloid leukemia

目的 探讨淀粉样前体蛋白（APP）基因在急性髓系白血病（AML）细胞的表达及其生物学行为.方法 应用实时定量PCR方法（2-ΔCt）检测85例初诊AML和20例非恶性血液病患者（对照）骨髓细胞APP mRNA的表达,并研究其表达水平对AML患者的临床特征和治疗反应的影响 应用实时定量PCR和Western blot方法检测APP基因和蛋白在AML细胞株的表达,并与AML原代细胞亚型的结果相比较 化学合成针对APP基因的小干扰RNA（siRNA）,利用脂质体转染HL-60细胞,下调APP基因表达.RNA干扰24、48、72 h后,采用MTT法及锥虫蓝拒染细胞计数检测细胞增殖 瑞特-姬姆萨染色观察细胞形态 PI染色流式细胞术分析细胞周期 Annexin V/PI双染色流式细胞术及Hoechst染色检测细胞凋亡 RNA干扰48 h,Western blot法检测凋亡相关蛋白NF-κB、bcl-2和caspase-3的表达 MTT法检测细胞对阿霉素的敏感性.结果 APP mRNA表达在AML亚型间差异有统计学意义（P=0.019）,伴t（8 21）的M2b表达最高（中位值0.1080）,其次是AML-未定型（0.0467）、M3（0.0266）、M2a（0.0221）、M4a（0.0167）、M5b（0.0151）、M4b（0.0025）,两两比较分析显示,M2b患者APPmRNA表达显著高于M5b患者（P=0.000）.APP mRNA表达水平对AML亚型以外患者临床特征及治疗反应无显著影响（P＞0.05）.Kasumi-1细胞APP基因表达显著高于U937细胞（P＜0.05）,与AML各亚型原代细胞中的结果相一致.APP-siRNA转染HL-60细胞后,其APP mRNA表达下凋64%,在培养24、48及72 h后检测HL-60细胞增殖、分化、凋亡、细胞周期及对阿霉素敏感性均无明显变化（P＞0.05）.结论 伴t（8 21）异位的M2型白血病高表达APP mRNA,单核细胞白血病低表达APPmRNA.APP基因表达沉默对HL-60细胞的生物学作用无显著影响.

Objective To investigate the expression of amyloid precursor protein （APP） gene in acute myeloid leukemia（AML） and its biological behaviour in AML cells. Methods The expressions of APP mRNA in 85 AML and 20 nonmalignant hematological diseases patients （as control） were measured by realtime PCR. The expression of APP in AML cell lines was also examined by real-time PCR and Western blot and the results were compared with those in their original subtypes. Small interfering RNAs（siRNAs） targeting APP gene were synthesized and transfected into HL-60 cell by lipofectamine 2000 for 24 h, 48h and 72 h. Cell growth was measured by trypan blue dye exclusion and MTT, differentiation by Wright-Giemsa staining, cell cycle by PL/RNase staining, apoptosis by Annexin V/PI and Hoechst33342 staining. Apoptosisrelated protein NF-κB, bcl-2 and Caspase-3 were detected by Western blot after siRNAs transfection for 48 h.Sensitivity to adriamycin was measured by MTT. Results The expression of APP mRNA among AML subtypes differed significantly （ P = 0. 019 ）, the highest expression subtype was M2 with t （ 8 21 ） （ median 0. 1080）, followed in order by AML-undefined （ 0. 0467 ）, M3 （ 0. 0266 ）, M2a （0.0221 ）, M4a （ 0. 0167 ）,M5b （0.0151）, and M4b （0.0025）. APP expression had no significant effect on AML clinical characteristics excepting for subtypes. The expression of APP in Kasumi-1 cells was significantly higher than that of U937cells （ P 〈0.05 ）, which was in agreement with APP expression in their original AML subtypes. After siRNAs transfection for 24 h, 48 h, and 72 h, no significant difference in proliferation, differentiation, apoptosis,cell cycle and sensitivity to adriamycin was detected between interfering group and control groups（ P 〉 0.05 ）.Conclusions The APP mRNA expression was highest in M2 with t（8 21 ） and lowest in M5b. Down-regulation of APP expression has no significant effects on biological behaviour of HL-60 cells.