实时荧光定量PCR检测急性髓系白血病miR-125b的表达及临床应用  

Detection and clinical application of miR-125b in acute myeloid leukemia by real-time fluorescence quantitative PCR

作　　者：王语欣 杨冬琴 周静东[1] 林江[2] 姚东明 杨静[1] 钱震[1] 杨磊[1] 钱军[1] 

机构地区：[1]江苏大学附属人民医院血液科,江苏镇江212002 [2]江苏大学附属人民医院中心实验室,江苏镇江212002

出　　处：《分子诊断与治疗杂志》2017年第5期334-340,共7页Journal of Molecular Diagnostics and Therapy

基　　金：国家自然基金项目(81270630);江苏省六大人才峰会项目(2015-WSN-115);镇江市社会发展项目(SH2015058);江苏大学临床医学科学发展基金会项目(JLY20140018)

摘　　要：目的采用实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,q PCR)法检测急性髓系白血病(acute myeloid leukemia,AML)中微小RNA-125b(micro RNA-125b,mi R-125b)转录本含量,初步评价其在AML临床诊断中的意义。方法建立扩增mi R-125b转录本的SYBR Green I染料法q PCR,对mi R-125b转录本克隆质粒进行扩增,评价其在AML诊断中的特异性、重复性和灵敏性。并对69例AML患者的骨髓单个核细胞标本进行初步检测并评价其临床相关性。结果该方法能定量检测骨髓标本中mi R-125b的表达水平,熔解曲线呈单峰,在108~103copies/μL范围内有良好的线性关系(R2=0.999)并且检测重复性良好。结果显示69例AML患者中mi R-125b表达水平明显高于25例对照组,差异有显著统计学意义(P=0.001)。在初发AML的不同亚型中,M3型的mi R-125b表达水平高于其他亚型,差异有统计学意义(P<0.05)。mi R-125b表达水平与非M3型AML患者的年龄、性别、外周血白细胞计数、血小板计数、血红蛋白含量、FAB分型、核型分组之间均无相关性(P>0.05)。4例初诊AML患者在获得完全缓解后mi R-125b表达水平均较治疗前有所降低。结论所建立的实时荧光定量PCR方法能敏感、特异地检测骨髓单个核细胞标本中mi R-125b的表达水平,为进一步临床应用研究奠定了方法学基础。mi R-125b异常高表达是AML中的一个常见分子事件,检测其表达可能有助于AML患者的辅助诊断和疾病状态监测。Objective To evaluate the method of real-time quantitative polymerase chain reaction（q PCR） for detecting miR-125 b transcript in patients with acute myeloid leukemia（AML） and further investigate the characteristics and clinical implication of miR-125 b in AML. Methods The reaction system and reaction condition of q PCR with SYBR Green I was established to detect the expression of miR-125 b to evaluate its specificity, repeatability and sensitivity in AML diagnosis. Afterwards, the samples of bone marrow mononuclear cells from 69 AML patients were evaluated. Results The method can detect the expression level of miR-125 b in AML patients. The melting curve showed a single peak, the PCR products was specific, a good linear relationship in the range of 108copies/μL to 103copies/μL（R2=0.999）, and the detection was repeatable. The expression level of miR-125 b in 69 AML patients was significantly higher than those in 25 controls（P=0.001）. In different subtypes of AML, the expression level of miR-125 b in M3 was higher than that in other subtypes, and the difference was statistically significant（P0.05）. There was no correlation between the expression of miR-125 b and the age, gender, peripheral blood leucocyte counts, platelet count,hemoglobincontent, FAB classifications and karyotypes（P0.05） in non-M3 AML patients. The levels of miR-125 b expression of 4 patients in the observation group decreased after complete remission. Conclusions The SYBR Green I q PCR for miR-125 b was a sensitive, reliable quantitative assay, and it can be used in the diagnosis of AML and the disease therapy evaluation. The increased-expression of miR-125 b may be a common molecular event in AML, and detecting its expression level could be used for auxiliary diagnosis and monitoring disease.

关 键 词：实时荧光定量PCR miR-125b 急性髓系白血病 

分 类 号：R730.43[医药卫生—肿瘤] R733.71[医药卫生—临床医学]