实时荧光定量PCR测定人肿瘤坏死因子相关凋亡诱导配体基因表达

目的 建立检测人肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因表达含量的实时荧光定量逆转录(FQ-RT)-PCR法,探讨其在健康对照者、乙型病毒性肝炎肝硬化患者及原发性胆汁性肝硬化(PBC)患者外周血单个核细胞(PBMC)中的表达状况.方法 设计TRAIL基因的特异性引物和TaqMan-MGB探针,以β-actin基因为内参,RT-PCR扩增目的 片段.胶同收纯化目的 片段,构建克隆载体作为定最模板,在ABI Prism7000型荧光定量分析仪上进行扩增,建立标准曲线;同时检测30名健康对照者、30例PBC患者及25例乙型病毒性肝炎肝硬化患者外周血PBMC中TRAIL基因的荧光强度和循环阈值,计算样本中TRAIL基因的起始拷贝数.结果 FQ-RT-PCR检测TRAIL mRNA的线性范围为103-106拷贝/ug RNA(r=-0.997);高浓度样本批内和批间变异系数(CV)分别为5.6%和6.3%.低浓度样本批内及批间CV分别为12.5%和14.6%.PBC患者TRAIL mRNA表达量为(3.3±2.5)×105拷贝ug RNA],高于健康对照组(0.5±0.2)×105拷贝/ug RNA],两组差异有统计学意义(t=5.994,P<0.01);乙型病毒性肝炎肝硬化患者TRAIL mRNA的表达(2.1±0.9)×105拷贝/ug RNA]亦高于健康对照组,且差异有统计学意义(t=8.536,P<0.01),而两疾病组间差异无统计学意义(t=1.988,P>0.05).结论 成功建立检测TRAIL mRNA的FQ-RT-PCR法,并初步发现TRAIL mRNA在PBC及乙型病毒性肝炎肝硬化患者外周血PBMC中表达升高,这将对肝硬化肝损伤机制研究提供新的思路.

Detection of TNF-related apoptosis inducing ligand gene expression by real-time fluorescent quantitative method

Objective To establish a real time fluorescent quantitative revers transcripatase PCR(FQ-RT-PCR) method to detect the expression level of TNF-related apoptosis inducing ligand (TRAIL) mRNA in peripheral blood mononuclear ceils (PBMC) and determine its expression level in healthy donors, HBV-caused cirrhosis patients and PBC ones. Methods Specific primers and Taqman-MGB probe were designed and β-actin was used as endogenous control. The amplified fragment was obtained by RT-PCR. The quantitative template was constructed and then the fluorescent intensity was documented on the ABI Prism7000 analyzer. The standard curve was established, according to which, the TRAIl. mRNA levels in 30 healthy individuals, 30 patients with primary biliary cirrhosis (PBC) and 25 ones with HBV-caused cirrhosis were calculated automatically by software after the values of cycle threshold (Ct) were detected continuously during amplification. Results The linear detection range of the assay for TRAIL gene was 103 - 109 copies/ ug RNA ( r=-0.997). The coefficients of variation of both intra-and inter-assay reproducibility for high concentration samples were 5.6% and 6. 3% , respectively, and those for low concentration samples were12.5% and 14. 6%. The TRAIL mRNA expression level in PBC patients was (3.3±2.5)×105copies/ugRNA] significantly higher than that of healthy control (0.5±0.2)×105 copies/ug RNA ] (t=5.994,P <0.01). TRAIl. mRNA level of HBV-caused cirrhosis patients (2.1±0.9)×105 copies/ug RNA] wasalso significantly elevated (t=8.536, P<0.01). However, the difference between these two diseased groups had no significance. Conclusion We have successfully set up a FQ-RT-PCR method for detecting TRAIL gene expression and found that its expression levels of peripheral blood mononuelear cells in PBC and HBV caused cirrhosis patients are elevated, which provides a new insight into mechanism study of liver injury caused by cirrhosis.