TRAIL mRNA实时荧光定量方法的建立及对慢性乙肝患者外周血单个核细胞检测的意义

Development of Real-Time Fluorescent Quantitative Method for Detection of TRAIL mRNA in Peripheral Blood Mononuclear Cell and Clinical Significance in Patients with Chronic Hepatitis B

目的：建立肿瘤坏死因子相关凋亡诱导配体（TRAIL）mRNA的实时荧光定量PCR检测方法,探讨慢性乙型肝炎（CHB）患者外周血单个核细胞（PBMCs）中TRAIL mRNA与HBV载量的关系及TRAIL在HBV感染后肝损伤中的作用。方法：自行建立检测TRAIL mRNA的TaqMan实时荧光定量RT-PCR法,对58例慢乙肝患者PBMCs中TRAILmRNA进行测定,采用夹心酶联免疫吸附法（ELISA）分析其血清可溶性TRAIL（sTRAIL）水平,实时荧光定量法检测PBMCs中HBV DNA含量,与肝功能相关指标进行相关性分析。结果：各型慢性乙型肝炎患者外周血单个核细胞TRAILmRNA表达水平高于健康者（t=28.311,P〈0.001）,但各组间差异显著性,与PBMCs中HBV载量无相关性。CHB轻度、CHB中度、重度-慢性重型肝炎、LC患者血清sTRAIL水平比健康者sTRAIL水平降低（t=0.801,P=0.430;t=2.315,P=0.027;t=2.672,P=0.013;t=5.085,P=0.000）,各型慢性乙型肝炎之间差异无统计学意义,与白蛋白（Alb）呈正相关（r=0.426,P=0.002）,与TBil、ALT及PBMCs中HBV载量无相关性。结论：慢性乙肝患者PBMCs中TRAILmRNA表达水平增加,血清sTRAIL水平降低,血清sTRAIL与Alb呈正相关,提示外周血单个核细胞膜结合型TRAIL增加,经TRAIL参与的肝细胞损害增加。

Objective：To establish the real-time fluorescent quantitative polymerase chain reaction（FQ-RT-PCR）for detecting TNF-related apoptosis inducing ligand（TRAIL） mRNA,and to explore the relationship between the levels of serum soluble TRAIL（sTRAIL）,TRAIL mRNA and the loads of HBV in the PBMCs of patients with chronic hepatitis B（CHB） and their roles in the liver damage.Methods：The mRNA expression of TRAIL and the loads of HBV in the PBMCs from 58 CHB patients were examined by real-time fluorescence quantitative polymerase chain reaction. The serum sTRAIL was examined by ELISA.The levels of TRAIL mRNA and sTRAIL were compared with the baseline levels of 20 healthy controls,and correlation analysis were performed with ALT,Alb,TBil and Loads of HBV in PBMCs.Results：The mRNA expression of TRAIL in PBMCs in CHB patients was much higher than the healthy controls（t=28.311,P〈0.001）,had no significant difference between various clinical types of hepatitis B,and had no correlation with the loads of HBV in the PBMCs. The serum levels of soluble TRAIL in mild,moderate,severe CHB and liver cirrhosis patients were lower than the healthy controls（t=0.801,P=0.430;t=2.315,P=0.027;t=2.672,P=0.013;t=5.085,P=0.000）,had no significant difference between various clinical types of hepatitis B.The level of sTRAIL was positively correlated with serum albumin（r=0.426,P=0.002）,had no relationship with TBil,ALT and the loads of HBV in the PBMCs.Conclusion： The mRNA expression of TRAIL in PBMCs in CHB patients is up-regulated.The serum level of sTRAIL is downregulated.The serum level of sTRAIL positively correlates with serum Alb.These findings suggests that the membrane bound TRAIL expression on PBMCs in CHB patients is up-regulated and the hepatocellular damage is increased by TRAIL pathway.