Objective of this study was to establish a SYBR Green Ⅰ real-time reverse transeription-polymerase chain reaction (RT-PCR) for quantitative detecticon of WT1 gene mRNA in children with acute myeloid leukemia (AML) and investigate its clinical significance. SYBR Green Ⅰ real-time RT-PCR was used to quantitatively detect the mRNA expression of WT1 gene in 30 newly diagnosed AML patients, 12 cases of remission (30), 18 relapsed patients and 30 cases of normal bone marrow cell morphology , and dynamically to detect the expression of WT1 gene in 20 newly diagnosed AML children. ABL served as internal reference gene, and the 2-ΔΔct method was used to calculate therelative expression. The results showed that ( 1 ) the expression of WT1 gene in newly diagnosed AML children was higher than that of the normal controls and the patients with remission (p ＜0.001 ); there were no significant difference of WT1 gene expression between AML patients with remission and normal controls (p ＞ 0.05 ), which were same as in relapsed patients and newly diagnosed patients (p ＞ 0.05); (2) WT1 gene in 20 newly diagnosed AML children highly expressed before the children were initially treated, decreased when they were complete remission, then expression increased again when their AML relapsed. The WT1 gene expression level began to rise in 5 cases before clinical relapse at 5 - 7 months; ( 3 ) the complete remission rate (CR) and 3 year overall survival (OS) did not show significant difference between the WTl-positive group and negative group when dinamically monitoring WT1 gene expression of 20 newly diagnosed children with AML. 3-year OS of WT1-positive group at the 22 - 30 days after initial treatment was significantly lower than that of the negative group (p ＜0.05). It is concluded that SYBR Green Ⅰ real-time RT-PCR is a rapid, efficient, sensitive and specific method. WT1 gene in AML chiidhood plays a role of cancer-promoting. The change of WT1 gene expression level contributes to evaluate the therapeutic efficacy, detect the minimal residual diseases and analyze the prognosis.%本研究建立实时荧光定量RT-PCR方法检测儿童急性髓系白血病WT1基因(AML)mRNA的表达并探讨其临床意义.采用SYBR Green I实时荧光定量RT-PCR方法,检测30例初治AML患儿、12例缓解期患儿(30份)、18例复发患儿,30例细胞形态学正常人的骨髓标本中的WT1基因的表达水平,并动态检测20例初治患儿WT1基因的表达.以ABL为内参照,采用2(-△△ct)法计算其相对表达量.结果表明:①初治AML患儿WT1基因的表达高于正常对照组和缓解组(P<0.001);缓解AML患儿WT1基因的表达与正常对照组的差异无统计学意义(p>0.05);复发AML患儿WT1基因的表达与初治组的差异无统计学意义(p>0.05);②动态监测儿童AML 20例显示,初治时WT1高表达,完全缓解后WT1表达水平下降,复发时WT1表达水平明显升高.20例中5例在临床复发前3-7个月表达水平开始上升.③动态检测的20例患儿初治时WT1阳性组和阴性组完全缓解率(CR)、3年总生存率(OS)无显著差异(p>0.05),治疗后第22-30天的WT1阳性组3年OS显著低于阴性组(p<0.05).结论:SYBR Green I实时荧光定量RT-PCR方法是一种快速有效,灵敏度高,特异性好的方法.WT1基因在儿童急性AML中表现为促癌基因的作用,WT1基因表达水平的变化有助于疗效评价,微小残留病的检测和预后判断.
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