人转化生长因子βlmRNA定量检测在肝纤维化病人中的应用研究  

Application of quantitative reverse transcription—polymerase chain reaction for transforming growth factor β1 in patients with hepatic fibrosis

作　　者：丁荣梅[1] 吕建新[1] 

机构地区：[1]温州医学院检验医学院细胞与分子医学研究所,浙江温州325035

出　　处：《检验医学教育》2004年第3期46-49,共4页

摘　　要：目的：建立实时定量RT—PCR检测人转化生长因子β1(transforming growth factorβ1，TGF-β1)的方法，对慢性肝炎肝纤维化患者外周血单个核细胞(peripheral blood mononuclear cell，PBMC)中TGF-β1 mRNA进行定量检测。方法：在TGF-β1基因的外显子1和2之间设计一对引物和一条TaqMan—MGB探针；构建TGF-β1质粒克隆，以T7 RNA聚合酶体外转录合成带有目的片断的cRNA作为标准品，根据10倍系列稀释cRNA标准浓度对数和其循环域值(Cycle threshold，Ct)制作标准曲线，检测PBMC中TGF—β1 mRNA含量；并对本方法进行方法学评价。结果：重组质粒测序显示插入的片断为TGF—β1 mRNA特异性片断，成功的建立了实时定量RT—PCR检测TGF-β1 mRNA方法，灵敏度达6．81拷贝，线性范围为6．81～6．81×10^8拷贝，且重复性好，批内、批间变异系数分别为1．28％-2．27％和2．56％-2．61％，临床应用显示，27例肝纤维化病人PBMC中TGF-β1表达量为1．20×10^8(1．80×10^7～9．80×10^7)拷贝数／10^5细胞，显著高于正常对照组3．30×10^7(7．24×10^6～5．00×10^7)拷贝数／10^5细胞(P<0．05)。结论：实时定量RT-PCR检测TGF-β1 mRNA具有敏感、特异、快速、高效等特点，为TGF—β1 mRNA作为肝纤维化的诊断指标提供方法学依据。objective:To develop real-time quantitative reverse transcription-polymerase chain reaction(Q-RT-PCR) for detection and quantitation of transforming growth factor β1 (TGF-β1) mRNA in peripheral blood mononuclear cell(PBMC) of patients with hepatic fibrosis, which is caused by HBV. Methods: A pair of primers were designed to amplify a fragment of 102 bp between exonl and exon2, and a TaqMan-MGB probe modified with Fam at 5'-end and minor groove binder at its 3'-end was designed. The TGF-β1 recombined plasmid was constructed by conventional molecular biological techniques, then strand-specific cRNA standard was synthesized by in vitro transcription. The cRNA standard was used for quantitation of the mRNA by plotting a standard curve of cycle threshold (Ct) values versus standard 10-fold dilutions of cRNA of known concentrations. The assay was evaluated. Results: Sequence analysis indicated that the recombined plasmid contains the specific 102bp fragment of TGF-β1. The real time Q-RT-PCR detected as low as 6.81 copies of standard cRNA, the linear range was from 6.81 to 6.81×10~9 copies, and results of the Q-RT-PCR were highly reproducible the coefficient variation was 1. 28%-2.27% and 2.56%-2.61% respectively in intra and inter-assay. The patients wih hepatic fibrosis had significantly higher TGF-β1 mRNA levels of 1.20×10~8(1.80×10~7～9.80×10~7) copies /10~5 cell than those in healthy controls of 3.30×10~7(7.24×10~6～5.00×10~7) copies /10~5 cell (P<0.05). Conclutions: Real-time Q-RT-PCR for TGF-β1 mRNA is a sensitive, specific, rapid, and efficient method that eliminates the post-PCR processing steps. Quantitation of TGF-β1 using real-time Q-RT-PCR will have application in studies aimed at understanding the diagnosis of hepatic fibrosis.

关 键 词：人转化生长因子β1 MRNA 定量检测 肝纤维化 TGF-Β1 RT-PCR 病理学 

分 类 号：R575[医药卫生—消化系统]