摘要
目的建立荧光定量逆转录聚合酶链反应(FQRTPCR)方法,检测慢性肝病患者外周血单个核细胞(PBMC)中TGFβ1表达水平。方法构建TGFβ1质粒克隆,以T7RNA聚合酶体外转录合成带有目的片断的cRNA作为标准品,在PCR反应体系中加入与模板互补的TaqManMGB探针建立荧光定量RTPCR,检测PBMC中TGFβ1mRNA含量,并对本方法进行方法学评价;同时检测血浆TGFβ1水平。结果重组质粒测序证明插入的片断为TGFβ1特异性片断,荧光定量RTPCR检测TGFβ1mRNA的灵敏度达6.81拷贝,线性范围为6.81～6.81×108拷贝,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,慢性乙型肝炎中、重度患者及肝炎后肝硬化患者PBMC中TGFβ1mRNA含量及血浆TGFβ1水平均显著高于正常(P<0.05)。结论荧光定量RTPCR检测TGFβ1mRNA具有敏感、特异、快速等特点,为TGFβ1mRNA作为肝纤维化的诊断指标提供方法学依据。
Objective To develop fluorescence quantitative reverse transcription polymerase chain reaction （FQ-RT-PCR） for detection and quantitation of transforming growth factor β1 （TGF-β1） mRNA in peripheral blood mononuclear cell （PBMC） of patients with chronic hepatic disease. Methods The TGF-β1 recombined plasmid was constructed by conventional molecular biological techniques, and strand-specific cRNA standard was synthesized by T7 RNA polymerase in vitro transcription. The cRNA standard and a TaqMan-MGB probe were used for quantitation of the TGF-β1 mRNA, and the assay was evaluated. Moreover,the plasma TGF-β1 was detected by ELISA. Results Sequence analysis indicated that the recombined plasmid contains the specific 102bp fragment of TGF-β1. The FQ-RT-PCR could detect as low as 6. 81 copies of standard cRNA, the linear range was from 6. 81 to 6. 81× 10^9 copies, and the coefficient variation was 1.28%-2. 27% and 2. 56%-2. 61% respectively in intra and inter-assay. The levels of TGF-β1 mRNA in PBMC and plasma TGF-β1 of patients with chronic hepatic disease were significantly higher than that of healthy controls（ P 〈 0. 05 ）. Conclusion FQ-RT-PCR is a sensitive, specific and rapid method that eliminates the post-PCR processing steps. Quantitation of TGF-β1 mRNA using FQ-RT-PCR will have application in studies aimed at understanding the diagnosis of hepatic fibrosis.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2005年第8期840-843,共4页
Chinese Journal of Laboratory Medicine
基金
浙江省自然科学基金资助项目(No.302769
No.397510)
