定量RT-PCR检测PBC患者外周血中颗粒溶素的基因表达水平  

Measurement of granulysin gene expression levels on peripheral blood of the patients with primary biliary cirrhosis by real-time quantitative RT-PCR

作　　者：钱琤[1] 陈波[1] 陈孙孝[1] 周晔[1] 王燕[1] 谷明莉[1] 吴传勇[1] 蒋廷旺[1] 陈燕[1] 邓安梅[1] 仲人前[1] 

机构地区：[1]第二军医大学附属长征医院实验诊断科,全军临床免疫中心,上海200003

出　　处：《中国实验诊断学》2006年第11期1241-1244,共4页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金(30471616);上海市重大科技攻关基金(04DZ19116);上海市重点科研支撑条件项目(051409012);上海市青年科技启明星计划(QMX01423);上海市重点基础研究项目(05JC14052)资助

摘　　要：目的建立检测人颗粒溶素(granulysin,GNLY)mRNA含量的实时荧光定量RT-PCR的方法,并测定PBC患者外周血单个核细胞(PBMC)中GNLY的基因表达水平,探讨GNLY基因表达水平与原发性胆汁性肝硬化(PBC,primarybiliary cirrhosis)发生发展的关系。方法基于TaqMan荧光探针技术,构建质粒pET28a-GNLY,作为定量模板,建立实时荧光定量RT-PCR方法。用此方法测定了60例PBC、60例乙型肝炎肝硬化及60例健康对照者外周血中GNLY mRNA的含量。结果PBC患者GNLY mRNA的表达范围为5.35×107-4.61×109拷贝/μg RNA;健康对照者为9.28×105-7.32×107拷贝/μg RNA。PBC组GNLY mRNA的平均拷贝数显著高于健康对照组(P<0.01)和疾病对照乙型肝炎肝硬化组(P<0.001)。结论成功建立了人GNLY基因表达含量的荧光定量检测方法,PBC患者GNLY mRNA的含量显著高于健康对照组,GNLY表达与PBC的发生发展存在一定的关联性。Objective To establish a rcal-time quantitative reverse transcription-polymerase chain reaction（RT-PCR） method for detecting the expression of GNLY gene in peripheral blood mononuclear cells（PBMC） and explore the relationship between GNLY mRNA expression and primary biliary cirrhosis（PBC）. Methods Based on fluorescent TaqMan methology, a real-time quantitative RTPCR was set up. In this method, a cloning vector pET28a-GNLY was constructed as a standard plasmid. The GNLY gene expression levels in 60 patients with PBC ,60 patients with hepatitis B related liver cirrhosis and 60 healthy controls were measured by real-time RT-PCR. Results The GNLY mRNA copy number ranged from 5.35 × 10^7 - 4.61 × 10^9copy/μgRNA in PBC patients and 9.28 × 10^5 - 7.32 ×10^7 copy/μgRNA in healthy controls.The mean GNLY mRNA copy number in PBC group was signilieantly higher than that in healthy controls（ P 〈 0.01）and hepatitis B related liver cirrhosis group（ P 〈 0. 001 ）. Conclusion A real-tlme quantitative RT-PCR method for detecting the expression of GNLY gene in PBMC has been sueeeesfully established. The expression level level GNLY is increased in PBC and GNLY gene expression level are associated with the occurrence and progression in PBC.

关 键 词：PBC GNLY 逆转录-聚合酶链反应 

分 类 号：R575.2[医药卫生—消化系统]