实时定量RT-PCR检测急性早幼粒细胞白血病PML-RARa融合基因  

Detection of PML/RARa Transcripts in Acute Promyelocytic Leukemia by Real-time Quantitative Reverse Transcription Polymerase Chain Reaction

作　　者：郎丽[1] 李惠民[1] 刘华[2] 王义民[2] 张学美[3] 

机构地区：[1]云南省第二人民医院血液科 [2]昆明医学院第一附属医院检验科,昆明650031 [3]昆明医学院第一附属医院血液科,昆明650031

出　　处：《中国实验血液学杂志》2007年第4期714-719,共6页Journal of Experimental Hematology

基　　金：云南省第二人民医院资助;编号05A002C

摘　　要：本研究旨在建立用实时定量RT-PCR检测急性早幼粒细胞白血病(APL)PML-RARa mRNA的方法,探讨APLPML-RARa融合基因表达水平与疗效的关系。用RT-PCR扩增培养的NB4细胞的PML-RARa融合基因,取1μgNB4细胞的总cDNA进行10倍的梯度稀释,作为标准品,建立荧光定量RT-PCR方法,对方法的灵敏性、稳定性、重复性进行了测定。定量检测4例急性早幼粒细胞白血病(APL)患者治疗前后骨髓内PML-RARa融合基因转录本水平,并对1例经治疗完全缓解后又复发的患者PML-RARa转录本水平(拷贝数)进行动态监测。结果表明:用本研究建立的实时定量RT-PCR方法可检测出10-5μgNB4细胞cDNA中的PML-RARa融合基因,其重复性的CT值,管间、批间变异系数(CV)分别为2.1%、3.8%。4例患者初治骨髓PML-RARa基因转录本水平的分别为1884、5533、1803、4677拷贝,平均为3475拷贝。经ATRA+化疗治疗后其PML-RARa基因转录水平下降至40、135、79、229拷贝,平均为121拷贝。还有1例患者治疗前PML-RARa基因转录本水平为8600拷贝,经过4个月治疗,虽然此时患者处于完全缓解期,但其转录水平拷贝数仍有730。3个月后患者出现复发,转录本水平升至为11000拷贝。经过ATRA+化疗治疗后转录本水平又逐渐下降至1200拷贝。结论;建立的实时定量RT-PCR灵敏、重复性好。治疗后APL患者的PML-RARa融合基因转录本水平明显下降,复发时基因转录本水平又升高。融合基因表达水平的改变与临床疾病进展关系一致,这有助于监测白血病微小残留病,评价疗效及判断预后。Tihs study was purposed to establish a real-time quantitative reverse transcription polymerase chain reaction （RT-PCR） for detection of PML/RARa fusion gene in patients with acute promyelocytic leukemia （APL） and to explore the relationship between the expression level of PML/RARa fusion gene transcript and the clinical status or efficacy of the therapy in APL. The conventional RT-PCR was used to amplify PML/RARa gene from cultured NB4 cells. Standard curves were constructed by modified real-time PCR on standard template after 10-fold serial dilutions of cDNA of 1 μg NB4 cells. The sensitivity, stability and repeatability of this method was determined. The PML/RARa gene transcripts of bone marrows in 4 APL patients before and after treatment and in 1 APL patient relapsed after complete remission were dynamically detected by modified real-time quantitative RT-PCR. The results indicated that the sensitivity of real-time quantitative RT-PCR for detecting PML/RARa fusion gene was about 10^-5 μg cDNA from NB4 cells, the repeatability and reproducibility of this method were satisfactory, intra-and inter-assay coefficients of variation were 2.1% and 3.8%. The copy numbers of PML/RARa transcripte reflecting PML/RARa fusion gene expression level in 4 newly diagnosed patients with APL were 1884, 5533, 1803, 4677 and the median was 3 475. After ATRA ＋ chemotherapy copy numbers of PML/RARa transcript decreased to 40, 135, 79, 29, and mean was 121. Another patient＇s PML/RARa gene copy number was 8600 at diagnosis, the gene copy number was 730 after therapy for 4 months, although he was in complete remission, but copy number increased to 11 000 when APL relapsed 3 months later. The copy number efficiently reduced to 1200 after ATRA ＋ chemotherapy. It is concluded that the established realreal-time quantitative RT-PCR method is sensitive, reliable, accurate and repeatable. The efficiency of method was finally tested for patient samples, showing a PML/RARa transcript copy number in APL patients significantly decr

关 键 词：实时定量RT-PCR 急性早幼粒细胞白血病 PML/RARa融合基因 微小残留病灶 

分 类 号：R733.71[医药卫生—肿瘤] Q503[医药卫生—临床医学]