乙型肝炎肝硬化患者外周血单个核细胞Toll样受体2、4的表达

Expression of Toll-like receptor 2,4 in peripheral blood mononuclear cells from patients with hepatitis B virus related cirrhosis

目的探讨乙型肝炎（乙肝）肝硬化患者中Toll样受体（TLR）2、4表达与细胞因子的相关性。方法采用随机数字表法随机收集35例乙肝肝硬化患者与35例健康对照者的静脉抗凝血。ELISA检测血TNF-α含量。分离两组外周血单个核细胞（PBMC），流式细胞仪检测PBMC表面TLR2、4表达。实时荧光定量PCR检测每份PBMC中TLR2、4mRNA的表达水平。正态分布均数比较采用t检验和单因素方差分析，非正态分布数据采用Mann—Whitney U检验、Kruskal-Wallis H检验和Spearman相关分析。结果肝硬化组的血TNF-α水平明显高于对照组（33．52ng／L比6．07ng／L，Z=-6．584，P〈0．01），且随着Child—Pugh评分的增加而升高。肝硬化组PBMC中TLR2阳性细胞率明显高于对照组（20．65％比12．04％，Z=-4．458，P〈0．01），与血TNF—α水平存在正相关（r=-0．4483，P〈0．05），且随着Child—Pugh评分的增加而升高。两组PBMC中TLR4阳性率差异无统计学意义。肝硬化组PBMC中TLR2/还原型磷酸甘油醛脱氢酶（GAPDH）mRNA表达水平明显高于对照组（0．2342比0．0431，Z=-6．83，P〈0．01），与血TNF-α水平存在正相关（r=0．4111，P〈0．05），且随着Child—Pugh评分的增加而升高。两组PBMC中TLR4mRNA表达水平差异无统计学意义。结论肝硬化患者PBMC中TLR2表达水平明显增高，与血TNF-α水平及肝硬化的严重程度呈正比，但TLR4表达水平无明显变化，提示在乙肝肝硬化患者中，是TLR2而非TLR4在肝硬化的进展过程中具有重要作用。

Objective To explore the relationship among cytokine levels and Toll-like receptor （TLR） 2,4 in hepatitis B virus （HBV） related cirrhosis. Methods Heparin anticoagulated venous blood of 35 randomly selected HBV related cirrhosis and 35 healthy volunteers were collected aseptically. Plasma tumor necrosis factor （TNF）-α concentration was determined using enzyme-linked immunosorbent assay （ELISA）. Peripheral blood mononuclear cells （PBMC） were separated and stained with anti-TLR2,4 monoelonal antibodies, then analysed by flow cytometry. Total RNA was extracted from PBMC and TLR2,4 mRNA expression levels were evaluated by real-time quantitative polymerase chain reaction （PCR） using SYBR Green detection. Means of normal distribution were compared by t test and one factor analysis of variance. The data of abnormal distribution were analyzed using Mann-Whithey U test, Kruskal Wallis H test and Spearman correlation analysis. Results The plasma concentration of TNF-α in the cirrhotic patients was significantly higher than that in the healthy controls （33.52 ng/L vs 6.07 ng/L, Z=-6. 584,P〈0.01）, which was parellel with Child Pugh score. TLR2 positive rate in PBMC from the cirrhotic patients was significantly higher than that from the healthy controls （20.65% vs 12.04% , Z=-4. 458, P〈0.01）, which was positively correlated with plasma TNF-α level （r=-0. 448 3, P〈0.05）, and parellel with Child-Pugh score. Between the cirrhotic and healthy groups, there was no significant difference of TLR4 positive rate in PBMC. The mRNA expression level of TLR2/GAPDH in PBMC from the cirrhotic patients was significantly higher than the controls （0. 234 2 vs 0. 043 1, Z=- 6.83. P〈0.01）, which was positively correlated with plasmaTNF-α level （r=0.411 1,P〈0.05）, and parellel with Child Pugh score. Between the two groups, there was no significant difference of TLR4 mRNA expression level in PBMC. Conclusions The expression of TLR2 in PBMC from cirrhotic patients is significantly elevated, which is positively correlated with plasma TNF-α level and the severity of liver disease. The expression of TLR4 in PBMC from cirrhotic patients is unchanged. It suggests that TLR2 but not TLR4, plays an important role in the progression of liver cirrhosis.