实时荧光定量RT-PCR检测急性白血病患者骨髓HB-1的表达

Detecting HB-1 Expression Level in Bone Marrow of Acute Leukemia Patients by Real-Time Fluorescence Quantitative RT-PCR

目的:探讨急性淋巴细胞白血病(ALL)患者骨髓细胞HB-1基因的表达水平以及HB-1基因在监测ALL微小残留病(MRD)方面的意义。方法:建立实时荧光定量RT-PCR方法(Taqman探针),检测HB-1基因的表达水平,并对其灵敏度、特异性和重复性进行检测。应用此方法检测了急性淋巴细胞白血病183例次,急性髓系白血病(AML)70例,血液系统非恶性疾病52例和造血干细胞健康供者24例的骨髓细胞HB-1基因的表达水平,并分析33例急性B淋巴细胞白血病(B-ALL)患者HB-1基因的表达水平与疾病诊断、复发的相关性。结果:构建的实时荧光定量PCR方法的灵敏度达10-4水平,批间变异系数为6.79%,批内变异系数为4.80%。检测到HB-1基因特异性表达于B-ALL细胞:初发/复发B-ALL中HB-1基因表达的中位水平明显高于ALL完全缓解组、初发T-ALL、初发AML、血液系统非恶性病及健康骨髓供者(33.0%vs 0.68%、0.07%、0.02%、0.58%、0,P<0.01),而初发TALL、初发AM L、血液系统非恶性疾病和健康供者HB-1基因的表达水平无统计学差异(P>0.05)。B-ALL患者血液学缓解时HB-1基因水平明显下降至0.68%(0-7.99%),疾病复发时HB-1表达水平再次升高(共3例,HB-1分别为7.69%、8.08%和484.0%),其检测结果与流式细胞学检测结果变化一致。结论:HB-1基因特异性表达于B-ALL细胞,所建立的实时荧光定量RT-PCR法在检测骨髓细胞HB-1基因的表达水平方面具有很好的灵敏度、特异性和重复性。HB-1基因可作为B-ALL患者的临床检测、监测MRD和早期预测复发的分子学标志。

Abatract Objective： To investigate the expression level of HB-1 gene in patients with acute lymphoblastic leukemia（ ALL） and the significance of HB-1 gene in monitoring of minimal residual disease（ MRD）. Methods： The method of real-time fluorescence quantitative RT-PCR（ Taqman probe） was established to detect the expression levels of HB-1 gene; then the sensitivity,specificity and repeatability of this assay were evaluated and verified. The HB-1 gene expression levels in bone marrowof 183 cases of ALL,70 cases of acute myeloid leukemias（ AML）,52 cases of nonmalignant hematologic diseases and 24 healthy hematopoietic stem cell donors were detected. The correlation of HB-1 level with diagnosis and relapse was analyzed by detecting bone marrowsamples of 33 B-ALL. Results： The sensitivity of this assay reached the 10^-4 level. The coefficient of variation for inter-batch and inter-tube of HB-1 were 6. 79% and4. 80%,respectively. It was found that HB-1 gene specifically expressed in acute B lymphoblastic leukemia. The median expression levels of HB-1 gene in newly diagnosed and relapsed B-ALL patients were statistically significantly higher than those in ALL in complete remission（ CR）,newly diagnosed T-ALL,newly diagnosed AML,non-malignant hematologic diseases,and healthy hematopoietic stem cell donors（ 33. 0% vs 0. 68%,0. 07%,0. 02%,0. 58% and 0,respectively）（ P〈0. 01）. No statistical differences were found between newly diagnosed T-ALL,newly diagnosed AML,non-malignant hematologic diseases and healthy donors（ P〉0. 05）. The expression level of HB-1 gene declined sharply when B-ALL patients reached complete remission（ 0-7. 99%,with median level 0. 68%）,but increased when relapsed（ 7. 69%,8. 08% and 484. 0% in 3 relapsed samples）,which was in accordance with results of flowcytometry.Conclusion： HB-1 gene specifically expressed in acute B lymphoblastic leukemia cells. The established real-time fluorescence quantitative RT-PCR assay shows good sensitivity,specificity and repeatability,thus,can be used as a biological marker in the clinical detection,monitoring MRD and predicting of early relapse for B-ALL patients.