实时定量PCR检测46例初诊急性早幼粒细胞白血病患者PML/RARα mRNA的分子表达  

Detection of PML/RARα Gene Transcripts in 46 Newly Diagnosed Acute Promyelocytic Leukemia Patients by Real-time Quantitative Reverse-Transcription Polymerase Chain Reaction

作　　者：主鸿鹄[1] 刘艳荣[1] 秦亚溱[1] 李金兰[1] 常艳[1] 王亚哲[1] 单福香[2] 江滨[1] 陆道培[1] 

机构地区：[1]北京大学人民医院北京大学血液病研究所,北京100044 [2]北京市道培医院,北京100049

出　　处：《中国实验血液学杂志》2007年第1期1-5,共5页Journal of Experimental Hematology

摘　　要：本研究探讨实时定量PCR(Q-PCR)检测PML/RARαmRNA的方法学,并对46例初诊急性早幼粒细胞白血病(APL)患者骨髓标本进行检测。构建PML/RARα的bcr1型和bcr3型转录本以及内参照abl基因转录本的阳性标准品质粒;利用ABI Prism7500型Q-PCR仪对46例初诊APL患者和40例非APL患者骨髓标本进行检测,PML/RARαmRNA定量结果以校正比值(NQ)表示,NQ=PML/RARαmRNA拷贝数/ABLmRNA拷贝数;应用四色流式细胞术检测免疫表型。结果显示,Q-PCR结果的日间差和日内差平均变异系数(CV)分别为1.58%和0·88%。可重复敏感度为可以检测5copies/100ng RNA。40例非APL患者PML/RARαmRNA均为阴性。46例初诊APL患者PML/RARαmRNA表达量NQ中位值为0.450(0.084-1.082)。比较32例bcr1型和14例bcr3型两组患者的特征表明,PML/RARαmRNA NQ中位值分别为0.454(0.084-1.082)和0.386(0.151-0.848)(P>0·05)。形态学诊断M3v的患者比例分别为9.40%和48.96%(P<0.05);初诊时WBC中位数分别为2.15(0.2-59.6)和9·35(0.91-122.8)(P<0.05),而在性别、年龄、初诊时血红蛋白和血小板计数、骨髓中APL细胞比例、DIC指标等方面无差异。流式细胞仪术检测时,CD45/SSC射门情况下,APL细胞群分布可以分为两类:高侧向角(L-SSC,粗颗粒)和非高侧向角(NL-SSC,细颗粒)两类。bcr1型患者中85.70%表现为L-SSC,而bcr3型患者中64·29%表现为NL-SSC。结论:建立的Q-PCR方法稳定可靠,敏感度高;bcr1型和bcr3型APL患者的PML/RARαmRNA表达量无差异,bcr3型APL患者中形态学M3v比例和WBC数比bcr1型患者高;PML/RARα不同转录本类型和免疫表型以及细胞形态学之间有一定的相关性。In order to explore the application of real-time quantitative reverse-transcription polymerase chain reaction （Q-PCR） for detecting PML/RARα gene transcripts in patients with acute promyelocytie leukemia （APL） , the bone marrow samples from 46 newly diagnosed APL patients were collected for analysis. Three plasmids containing eDNA fragments of the bcr1-, bcr3-form PML/RARα and ABL control gene were constructed respectively. The ABI Prism 7500 Sequence Detection System using Taqman fluorogenie probes was used to quantify target gene. PML/RARα mRNA was detected by Q-PCR in 46 APL patients and 40 non-APL patients. The normalized quotient （NQ） of PML/RARα mRNA was calculated as followings： NQ = PML/RARα mRNA copy numbers/ABL mRNA copy numbers. Immunophenotype of acute promyelocytie leukemia was determined by four-color flow eytometry. The results showed that the coefficients of variation （CV） of inter-day assay and intra-day assay by using Q-PCR were 1.58% and 0.88% respectively. Q-PCR could detect reprodueibly 5 copies of target gene per 100 ng RNA and no pseudopositive results were found. The median NQ of PML/RARα mRNA was 0.450 （0. 084 - 1. 082） in 46 APL patients. There was no indication of any correlation of PML/RARα mRNA type with age, sex, hemoglobin, platelet count, percentage of promyelocytes in bone marrow detected by morphology or flow eytometry,PML/RARα NQ, or signs of clinically diagnosed coagulation/bleeding distich. Compared with bcr1-form cases, bcr3-form cases had more M3v phenotype （42.9% vs 9.4%, P=0.015） and higher WBC count （9.35 ×10^9/L vs 2.15 × 10^9/L, P =0.038 ）. APL cells could be classified into large side scatter population （L-SSC） and non-large side scatter population（NL-SSC） in CD45/SSC histogram of flow cytometry. 87. 50% patients with bcrl-form showed L-SSC phenotype and 64.29% patients with bcr3-form showed NL-SSC phenotype. It is concluded that a sensitive Q-PCR method is established. The median NQ of PML/RARα mRNA was 0.450 in newly d

关 键 词：白血病 急性早幼粒细胞白血病 实时定量PCR PML/RARΑ 

分 类 号：R733.71[医药卫生—肿瘤] R466.1[医药卫生—临床医学]