慢性乙型重型肝炎患者外周血树突状细胞核因子-κB及其激活因子的表达及意义

Expression of nuclear factor- κB and its regulated cytokines in dendritic cells in patients with chronic severe hepatitis B and its clinical significance

目的 探讨慢性乙型重型肝炎（CSHB）患者外周血单个核细胞衍生的树突状细胞核因子（NF）-κB、肿瘤坏死因子（TNF）α、白细胞介素（IL）-6与疾病严重程度及预后的相关性.方法 采集37例CSHB患者、20例慢性乙型肝炎（CHB）患者和20例健康对照者的外周血,分为CSHB组、CHB组与对照组并分别用密度梯度离心法分离外周血单个核细胞,用免疫磁珠细胞分选法获得纯化的单核细胞,体外培养6 d为未成熟的树突状细胞,用聚肌胞刺激48 h为成熟的树突状细胞（mDC）.用实时定量PCR检测mDC NF-κB p50表达;用酶联免疫吸附试验测定培养mDC上清液中TNF α、IL-6的分泌水平.两组间比较采用t检验,3组间比较采用One-Way ANOVA分析法,相关分析采用直线相关分析法.结果 3组研究对象mDC表达NF-κB p50,表达量CSHB组为2.63±0.94、CHB组为2.03±0.39、对照组为1.41±0.40,3组比较,F=19.01,P〈0.01,差异有统计学意义.mDC分泌TNF α,CSHB组、CHB组、对照组分别为（15 317.69±4124.90）pg/ml、（9670.29±3654.68）pg/ml、（6547.43±1027.20）pg/ml,3组比较,F=45.77,P〈0.01,差异有统计学意义; IL-6分泌量分别为（1423.78±375.14）pg/ml、（862.68±93.68）pg/ml、（567.26±167.04）pg/ml,3组比较,F=67.60,P〈0.01,差异有统计学意义.3组研究对象mDCNF-κB p50表达与TNF α、IL-6水平呈正相关（r=0.52,P〈0.01;r=0.65,P〈0.01）.CSHB组mDC表达TNF α、IL-6与凝血酶原活动度呈负相关（r=-0.41,P=0.01;r=-0.40,P=0.01）,与ALT、总胆红素、HBV DNA无相关性（r=-0.03,P=0.85,r=0.01,P=0.93,r=0.01,P=0.95;r=-0.09,P=0.58,r=0.16,P=0.34,r=0.09,P=0.59）.CSHB 存活组与死亡组mDC表达TNF α、IL-6差异无统计学意义（t=0.42,P=0.67;t=0.76,P=0.45）.结论 CSHB患者树突状细胞衍生的NF-κB及其激活产物TNF α、IL-6表达增加,可能与CSHB疾病严重程度相关.

Objective To investigate the correlation of nuclear factor- κ B （NF- κ B） level and its regulated cytokines in monocyte-derived dendritic cells （MoDC） with illness severity and prognosis in patients with chronic severe hepatitis B （CSHB）. Methods Peripheral blood mononuclear cells （PBMC） were isolated by Ficoll density gradient separation from 37 patients with CSHB, 20 chronically HBV-infected patients, and 20 normal controls. Monocytes were acquired using immunomagnetic anti-CD14-beads. Next,monocytes were induced into immature DC （iDC） in vitro. On day six, polyI：C was added to induce DC maturation. Then mature DCs （mDCs） were collected at 48h after polyI：C treatment to test the expression of NF- κB p50 by real time PCR. The supernatants were also collected respectively. The expression of TNF α and IL-6 in the supernatants were measured by ELISA. Results The expression of NF- κB p50 in mDCs of patients with CSHB was the highest in three groups （F = 19.01, P 〈 0.01）. The secretion of TNF α and IL-6 in mDCs from group CSHB was extremely high when compared with the other two groups, the secretions of TNF α in groups CSHB, CHB and NC were（15317.69 ± 4124.90） pg/ml, （9670.29 ± 3654.68） pg/ml and （6547.43 ± 1027.20） pg/ml （F = 45.77, P 〈 0.01） respectively, and the productions of IL-6 in groups CSHB,CHB and NC were （1423.78 ± 375.14） pg/ml, （862.68 ± 93.68） pg/ml and （567.26 ± 167.04） pg/ml （F=67.60, P 〈 0.01）, respectively. NF- κ B p50 showed significant correlations with TNF α （r= 0.52, P 〈 0.01）and IL-6 （r = 0.65, P 〈 0.01） in mDCs. Furthermore, the secretions ofTNF α and IL-6 in mDCs from group CSHB were negatively associated with PTA （r = -0.41, P = 0.01; r = -0.40, P = 0.01）, but not with ALT, TBil and virus loads （r= -0.03, P = 0.85, r = 0.01, P = 0.93, r = 0.01, P = 0.95; r = -0.09, P = 0.58, r = 0.16, P =0.34, r = 0.09, P = 0.59）. No significant difference found in the expression of TNF α and IL-6 between the survival subgroup and the death subgroup in group CSHB （t = 0.42, P = 0.67; t = 0.76, P =0.45）. Conclusions The expression levels of NF- κ B and its regulated cytokines in monocytederived DCs in patients with chronic severe hepatitis B were up-regulated and perhaps associated positively with the illness severity.