DIG掺入PCR半定量检测AML中Flt3基因的表达

THE EXPRESSION OF FLT3 GENE IN AML DETECTED BY SEMI-QUANTITATIVE RT-PCR WITH INCORPORATION OF DIG

目的 :观察 Flt3基因在 AML(急性髓细胞性白血病 )病人骨髓中的表达水平及正常对照骨髓、外周血表达水平的变化及其与病情、疗效的关系。方法 :应用血细胞 Percoll不连续密度梯度分离法分离出骨髓中恶性细胞 ,采用 DIG掺入半定量 RT- PCR,检测 Flt3基因在 2 2例 AML初治病人、5例 AML CR病人的骨髓、10例正常人和非恶性血液病病人 13例外周血、17例骨髓以及 5种白血病细胞株中的表达水平。结果 :5种白血病细胞株中测得 Flt3表达值分别为 NB40 .97,HL- 6 0 1.1,KG- 10 .96 ,K5 6 2及 U937均为 0 ;正常人骨髓为 0 .18～ 0 .45 ,平均 0 .2 2 ;正常人外周血未见表达 ;AML CR骨髓表达为 0 .2～ 0 .6 5 ,平均 0 .2 8。初治 AML 骨髓中 Flt3基因 m RNA表达率为 10 0 % ;其表达水平为 0 .75～ 1.2 4,平均为 0 .96 ,为正常骨髓的 4倍。结论 :在 AML中 Flt3基因为高水平表达 ,主要来源于白血病细胞 ,其检测水平可作为诊断疾病、判断预后的一项灵敏指标。

Objective: To observe the expression of flt3 gene in AML bone marrow. In order to explore the relation between the expression of flt3 gene and the occurrence or development and / or prognosis of these diseases. Methods : The separation method of the percoll discontinuous density gradient was applied to isolate the blast cells in above samples. Semi quantity RT PCR by means of incorporation of digoxigenin was used to detect the mRNA expressions of Flt3 in 22 primary AML blasts,5 AML CR, their counter parts from bone marrow of 17 patients, from white cells of peripheral blood of non malignant patients and 10 normal adults and 5 leukemia cell lines. Results : The expression level of the Flt3 mRNA in five leukemia cell lines were as follows NB4 0.97,HL 60 0.96, KG 1 0.96, K562 0, U937 0; Normal it was 0.22 in bone marrow ,but in normal peripheral blood it was no expression ,AML CR was 0.28, the positive rate in primary AML blast was 100%;its level was four times than that of normal bone marrow. Conclusion: High level expression of Flt3 gene in AML is mainly originated from blasts ,therefore ,Flt3 gene expression could be used as a sensitive and convenient parameter for diagnosis and prognosis.