摘要
目的：建立一种血清循环B细胞特异性莫洛尼白血病毒插入位点1（ Bmi-1）信使核糖核酸（mRNA）直接逆转录-实时荧光定量聚合酶链反应（RT-qPCR-D）检测方法，并运用该方法对结直肠癌血清循环Bmi-1 mRNA水平进行分析，探讨其在结直肠癌诊断中的价值。方法方法学建立。提取结直肠癌HT29细胞株RNA，建立Bmi-1、泛素C （ UBC）、甘油醛-3-磷酸脱氢酶（ GAPDH） mRNAs检测标准曲线，计算扩增效率。利用RT-qPCR-D方法对血清、准备液混合液中Bmi-1 mRNA进行直接检测，通过熔解曲线评价试验的特异性，以及血清样本稀释观察方法的检测下限。并且，用该方法检测2008年1月至2009年1月山东大学齐鲁医院158例结直肠癌患者，其中Ⅰ期26例、Ⅱ期53例、Ⅲ期47例和Ⅳ期32例，以及53例结直肠镜检查正常者血清循环Bmi-1 mRNA水平，采用Kruskal-Wallis H、Mann-Whitney U检验比较各组Bmi-1 mRNA表达水平，受试者工作特征曲线（ ROC）分析其对结直肠癌的检测效能。结果 Bmi-1、UBC、GAPDH log值与定量循环（ Cq ）值间呈良好线性关系（R2＝0.990、0.990、0.99，P均＜0.001），扩增效率（E）分别为0.875、0.917、0.935。建立的RT-qPCR-D方法，检测Bmi-1、UBC、GAPDH mRNAs的熔解曲线峰值单一；检测下限可达1.25μl。运用该方法检测血清循环Bmi-1 mRNA水平，在结直肠癌Ⅰ期患者中为0.138（0.078-0.228），Ⅱ期患者中为0.163（0.067-0.287）、Ⅲ期患者中为0.217（0.072-0.267）、Ⅳ期患者中为0.273（0.139-0.419）以及对照者中为0.021（0.008-0.029），各组间比较差异有统计学意义（H＝89.5，P＜0.001）。结直肠癌各期组循环Bmi-1 mRNA水平明显高于对照组（U＝58.0、287、246、72.5，P均＜0.001）；循环Bmi-1 mRNA在Ⅳ期患者中的水平明显高于其他各期患者（U＝247、590、540，P＝0.008、0.020、0.035），而在Ⅰ期、Ⅱ期、Ⅲ期患者间比较，差异无统计�
Objective To establish a direct reverse transcription real-time fluorescence quantitative polymerase chain reaction （ RT-qPCR-D ） method for detecting serum circulating B cell-specific moloney murine leukemia virus integration site-1 （Bmi-1） mRNA, and analyze the levels of serum circulating Bmi-1 mRNA in colorectal cancer patients by using of this method for exploring its diagnosis value in colorectal cancer.Methods Methodology establishment.RNA was extracted from colorectal cancer HT 29 cell line, and detection standard curves of Bmi-1, ubiquitin C （ UBC）, glyceraldehyde-3-phosphate dehydrogenase （ GAPDH） mRNAs were established , then the amplification efficiencies were calculated.Bmi-1 mRNA level was directly detected in serum and preparation buffer mixture , then the specificity of assay was evaluated by melting curve, and detection limit was observed through diluted serum samples.The serum circulating Bmi-1 mRNA levels were detected by ELISA in 158 cases with colorectal cancer , of which there were 26 cases of tumor node metastasis （ TNM）Ⅰstage, 53 cases of TNMⅡ, 47 cases of TNMⅢ, 32 cases of TNMⅣand 53 cases of controls with normal colonoscopy collected from January 2008 to January 2009 in Qilu Hospital of Shandong University.Comparisons of groups were determined by applying Mann-Whitney U test or Kruskal-Wallis test, and receiver operating characteristic （ ROC） curves were established to illustrate the diagnostic performance.Results The log values of Bmi-1, UBC and GAPDH showed good linear correlations with quantification cycle （Cq） values（R2 =0.990, 0.990, 0.991, all P 〈0.001）, and the amplification efficiencies were 0.875, 0.917 and 0.935, respectively.Using the established RT-qPCR-D method, the peak of melting curve of Bmi-1, UBC and GAPDH mRNAs were single, the detection limit was up to 1.25μl.The levels of serum circulating Bmi-1 mRNA detected by RT-qPCR-D were 0.138 （ 0.078-0.228 ） in colorectal cancer stage Ⅰ patients, 0.163（0.067 -0.287） instage �
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2014年第9期678-682,共5页
Chinese Journal of Laboratory Medicine
基金
国家自然科学基金（81271916,81301506）
高等学校博士学科点专项科研基金（20120131110055,20130131120067）
山东省自然科学基金（ZR2013HQ063）
关键词
结直肠肿瘤
逆转录聚合酶链反应
敏感性与特异性
Colorectal neoplasms
Reverse transcriptase polymerase chain reaction
Sensitivity and specificity
