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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 real-time fluorescence quantitative RT-PCR method for the determination of granulysin (GNLY) mRNA in peripheral blood mononuclear cells (PBMCs) of PBC patients and to determine the expression level of GNLY gene The relationship between the occurrence and development of primary biliary cirrhosis (PBC). Methods Based on TaqMan fluorescent probe technology, plasmid pET28a-GNLY was constructed and used as a quantitative template to establish a real-time fluorescence quantitative RT-PCR method. Using this method, the levels of GNLY mRNA in peripheral blood of 60 patients with PBC, 60 patients with hepatitis B cirrhosis and 60 healthy controls were determined. Results The expression of GNLY mRNA was 5.35 × 107-4.61 × 109 copies / μg RNA in PBC patients and 9.28 × 105-7.32 × 107 copies / μg RNA in healthy controls. The mean copy number of GNLY mRNA in PBC group was significantly higher than that in healthy control group (P <0.01) and in disease control hepatitis B cirrhosis group (P <0.001). Conclusion The fluorescent quantitative detection method of human GNLY gene expression was successfully established. The content of GNLY mRNA in PBC patients was significantly higher than that in healthy controls. The expression of GNLY was correlated with the occurrence and development of PBC.