摘要
目的:建立SYBR GreenⅠ实时定量RT-PCR方法检测白血病细胞中BMI-1 mRNA的表达并探讨其临床意义。方法:在TRIzol提取总RNA后,将mRNA逆转录成cDNA,再用SYBR GreenⅠ实时定量PCR方法检测BMI-1 mRNA在白血病细胞系(K562、HL-60、Daudi、Jurkat、Molt-4、U937和THP-1)、56例AML初治患者的骨髓ACDA抗凝标本和30例健康体检者的外周血EDTA抗凝标本以及6例细胞形态学正常的骨髓ACDA抗凝标本中的表达,扩增产物的特异性通过熔解曲线和凝胶电泳双重确认,以ABL为内参照,采用2-△△ct法计算其相对表达量。结果:①BMI-1 mRNA在白血病细胞系(K562、HL-60、Daudi、Jurkat、Molt-4、U937和THP-1)均存在表达,其中U937表达最高,而HL-60表达最低。②与对照组相比,BMI-1 mRNA在白血病细胞中的表达显著升高(P<0.01)。结论:①SYBR GreenⅠ实时RT-PCR是一种快速有效、灵敏度高、特异性好的定量检测BMI-1 mRNA的方法。②BMI-1参与白血病的发生,可能是一种新的肿瘤分子标记物。
Objective： To establish a SYBR Green I real-time reverse transcription-polymerase chain reaction RT-PCR） for quantitating human B-cell specific moloney leukemia virus insertion site 1 （BMI-1） mRNA and study Its expression in leukemic cells. Method： Total RNA was extracted with TRIzol and mRNA was transcribed reversely into cDNA. SYBR Green I real-time PCR was used to quantitate mRNA expression of BMI-1 and reference gene ABL in leukemic cell lines （K562, HL-60, Daudi, Jurkat, Molt-4, U937 and THP-1）, 56 samples of bone marrow cells from acute myeloid leukemia of initial presentation. For normal controls, 6 normal bone marrow samples and 30 normal peripheral blood samples were used. The specific amplified products were identified by gel electrophoresis with ethidium bromide staining, and the characteristic of their melting temperatures （Tm） were analysed by melting curves. Result： （1）BMI-1 mRNA was detected in all the leukemic cell lines in the study. U937 showed a high expression level and HL-60 showed a low expression level. （2）The relative expression level of BMI- 1 mRNA in leukemic cells and normal controls was 0. 864±0. 327 and 0. 102±0. 068, respectively （ P 〈0.01）. Conclusion：（1）SYBR Green I real-time RT-PCR is a rapid, sensitive and specific method to quantitate BMI-1 mRNA. （2）BMI-1 may contribute to leukemogenesis and may be a new tumor marker.
出处
《临床血液学杂志》
CAS
2009年第3期261-265,共5页
Journal of Clinical Hematology
基金
湖北省科技攻关项目(No:2005AA304B08)
