54例慢性乙型肝炎患者肝组织乙型肝炎病毒共价闭合环状DNA与血清HBsAg定量检测结果分析

目的 检测慢性乙型肝炎患者肝组织HBV共价闭合环状DNA (cccDNA)和血清HBsAg,分析两种定量指标之间及其与血清HBV DNA载量的相关性.方法 应用PSAD消化+滚环扩增+跨缺口实时荧光PCR方法,定量检测54例慢性乙型肝炎患者甲醛固定石蜡包埋肝组织HBV cccDNA水平;用化学发光试剂定量检测患者血清HBsAg.用Pearson检验及直线回归分析方法 对数据进行分析.结果 患者肝组织HBV cccDNA与血清HBsAg定量水平之间呈正相关(r=0.459,P＜0.01),但与血清HBV DNA载量相关性无统计学意义;血清HBsAg定量水平与血清HBV DNA载量呈正相关(r=0.328,P＜ 0.05),与病毒复制效率呈负相关(r=-0.373,P＜0.05).结论 慢性乙型肝炎患者肝组织HBV cccDNA载量与血清HBsAg定量水平相关,结合血清HBVDNA定量检测,可以更全面的反映HBV的复制水平,评价抗病毒疗效.     

慢性乙型肝炎患者血清、肝内HBV DNA定量及肝组织HBV DNA载量与肝组织损害程度的关系

目的 了解慢性乙型肝炎患者血清、肝内HBV DNA定量及肝组织HBV DNA载量与肝组织损害程度的关系.方法 经血清学及肝穿刺病理证实为慢性乙型肝炎患者38例,分为2组(HBeAg阳性组及HBeAg阴性组).采用ELISA法测定HBV-M、实时荧光定量检测血清及肝内HBV DNA定量、肝组织HBV DNA载量.结果 HBeAg阳性组血清、肝内HBV DNA定量及肝组织HBV DNA载量均高于HBeAg阴性组(P均<0.001);血清HBV DNA定量随肝脏纤维化程度加重而升高(P<0.01),但与肝脏炎症程度未见明显相关;肝组织HBV DNA载量则随肝脏炎症及纤维化程度加重而降低(P<0.001).结论 血清HBV DNA定量与肝组织纤维化程度呈正相关,肝组织HBV DNA载量则与肝组织的炎症及纤维化程度呈明显负相关.     

定量检测慢性乙型肝炎患者肝组织乙型肝炎病毒DNA的临床价值

目的探讨慢性乙型肝炎(CHB)患者肝内乙型肝炎病毒(HBV)DNA载量与血清HBV DNA、乙型肝炎病毒e抗原(HBeAg)水平的相关性及其在抗病毒治疗中的意义.方法41例HBeAg阳性CHB患者,在干扰素α和拉米夫定联合治疗前进行肝穿刺,取肝组织分别进行HBV DNA检测及组织学检查,据肝组织HBVDNA载量小于等于或大于104fg/cm3将其分为两组,治疗前及治疗期间监测其肝功能、血清HBeAg及HBV DNA情况.结果(1)肝组织HBV DNA载量高于血清HBV DNA载量(对数值4.081±1.127与3.163±1.010,t=2.218,P＜0.05),二者高度相关(r=0.840,t=4.322,P＜0.001);肝组织HBV DNA载量与血清HBeAg亦呈正相关(r=0.459,t=3.056,P＜0.005).(2)肝组织HBV DNA载量与肝组织炎症活动度呈反向关系(x2=3.874,P＜0.05).(3)治疗期间两组患者血清HBV DNA水平均明显下降,治疗前肝组织HBV DNA水平低者效果较好;治疗1年时HBeAg、抗-HBe血清转化率以肝组织HBV DNA水平低者为高(HBeAg转阴率68.4%与36.4%,x2=4.194,P＜0.05;抗-HBe阳转率73.7%与40.9%,x2=4.447,P＜0.05).结论肝组织HBV DNA水平较血清HBV DNA、HBeAg水平更能准确反映肝组织HBV DNA复制情况,且能间接反映机体的免疫状态,可作为抗病毒治疗适应证选择及疗效预测因子.     

慢性HBV感染者肝脏HBV cccDNA含量相关因素分析

目的探讨慢性HBV感染者肝组织中HBV cccDNA含量与血清病毒标志物、HBV DNA及肝脏病理分级的关系,为临床评价抗病毒治疗效果及疗程确定提供理论依据。方法以2007年5月-2008年2月住院的30例慢性HBV感染者为研究对象,应用实时荧光定量聚合酶链反应（RT-PCR）方法检测患者肝组织中HBV cccDNA、肝组织总HBV DNA（HBV tDNA）和血清HBVDNA,同时用化学发光免疫分析法检测HBsAg、HBeAg定量,分析感染者肝组织内HBV cccDNA与肝组织内HBV DNA、血清HBVDNA、HBsAg及HBeAg定量水平之间的关系,并比较肝组织中HBV cccDNA含量与肝脏病理炎症和纤维化分级的关系。采用Pear-son简单相关和Spearman等级相关法进行相关性分析。结果 30例慢性HBV感染者肝组织中均可检出HBV cccDNA,范围在3.15×103～1.06×107拷贝/mg;肝组织cccDNA定量与肝组织总HBV DNA定量呈正相关（r=0.375,P〈0.05）,与血清HBV DNA无相关性（r=0.174,P〉0.05）;肝组织中HBV cccDNA水平与血清HBsAg定量呈高度正相关（r=0.562,P〈0.001）,而与血清HBeAg定量无相关性（r=0.152,P〉0.05）。肝组织cccDNA定量与肝组织炎症活动度（G）及纤维化程度（S）无相关性（r=0.082,P〉0.05）。结论慢性HBV感染者肝组织内HBV cccDNA成稳定的中等水平复制;血清HBV DNA载量不能直接代表其肝组织中的HBV cccDNA水平;血清HBsAg定量可作为反映肝组织中HBV cccDNA水平的指标。     

慢性乙型肝炎患者肝组织乙型肝炎病毒cccDNA和血清病毒学标志物相关性的研究

目的探讨CHB患者血清病毒学标志物及肝组织、血清HBV DNA与肝组织HBVcccDNA的关系。方法应用实时荧光定量法进行20例CHB患者肝组织HBVcccDNA、肝组织及血清HBV DNA定量的测定,时间分辨荧光免疫分析法进行HBsAg、HBeAg、HBcAb等血清学标志物的测定,分析肝组织HBVcccDNA与肝组织HBV DNA、血清HBV DNA、HBsAg及HBeAg定量水平之间的关系。相关性分析采用Spearman相关性检验。结果所有患者肝组织中均能检测到HBVcccDNA,含量在7.63×10~4拷贝数/mg~9.46×10~8拷贝数/mg(对数值:7.55±1.08)之间;肝组织HBVcccDNA与肝组织HBV DNA呈显著正相关关系(r=0.859,P0.01),与血清HBV DNA呈显著正相关关系(r=0.640,P0.01);肝组织HBVcccDNA与HBeAg呈显著正相关(r=0.676,P0.01),与血清HBsAg无相关性(r=0.195,P0.05),与HBcAb呈显著负相关(r=-0.624,P0.01)。结论 CHB患者肝组织内HBVcccDNA呈稳定的中等水平复制;血清HBsAg定量尚不能作为反映肝组织中HBVcccDNA水平的指标,结合肝组织HBV DNA、血清HBV DNA等定量检测可以更全面反映HBV复制水平及患者病情评估。     

母亲血清乙型肝炎病毒载量及新生儿外周血游离母亲DNA与新生儿乙型肝炎病毒感染的关系

目的 探讨慢性HBV感染母亲血清HBV DNA载量及新生儿外周血中游离母亲DNA与新生儿HBV感染的关系.方法 等位基因特异性PCR及半套式PCR技术检测慢性HBV感染母亲及其新生儿外周血中游离母亲DNA.荧光定量PCR对母亲外周血血清中HBV DNA定量检测.数据采用阳性率、X2检验、Fisher精确概率等进行统计学分析.结果 筛选出36对母儿信息病例对,有26个新生儿外周血中检测到游离母亲DNA,占72.2%.母-胎DNA转移与新生儿HBsAg、HBV DNA阳性均无关(Fisher精确概率分别为0.278、1.000,均P>0.05),而与新生儿外周血单个核细胞(PBMC)HBV感染有关(Fisher精确概率＝0.026,P<0.05).母-胎DNA转移与母亲血清HBV DNA载量无关(X2=2.097,P>0.05),随着母亲血清HBV DNA载量的增加,其发生新生儿血清HBV DNA阳性的危险性呈增高趋势(总X2=62.21,P<0.05;趋势X2=58.46,P<0.05),而母亲血清HBV DNA载量与新生儿PBMC HBV DNA阳性无关(总X2=4.82,P>0.05).结论 母胎DNA转移与新生儿PBMC HBV感染有关,可能是新生儿HBV感染的一个因素.随着母亲HBV DNA载量增高,新生儿血清HBV DNA阳性的危险性越高.     

慢性乙型肝炎患者血清和外周血单个核细胞IL-18水平与HBV感染的关系

目的 初步探讨慢性乙型肝炎(CHB)患者血清和外周血单个核细胞(PBMC)白细胞介素-18(IL-18)水平与乙型肝炎病毒(HBV)感染的关系.方法 ELISA法检测HBV感染者(轻、中、重度三组)和正常组的血清与PBMC IL-18水平,同时检测血清AJJT/AST水平;荧光定量PCR检测各组患者血清和PBMC HBV DNA量,并分成HBV DNA病毒载量低、中、高三组.结果 CHB各组患者间血清和PBMC培养上清IL-18水平有显著差异(P＜0.05),由高到低依次为:重度组、中度组、轻度组;血清IL-18水平较对照组明显升高(P＜0.05),PBMC培养上清IL-18水平明显低于对照组(P＜0.05);不同血清病毒载量组,血清IL-18水平有显著差异(P＜0.05):由高到低依次为:低病毒载量组、中病毒载量组、高病毒载量组;PBMC内HBV DNA阴性组IL-18水平明显高于HBV DNA阳性组(P＜0.05);CHB患者血清IL-18水平与血清ALT/AST呈显著正相关.结论 IL-18可能参与了CHB的免疫应答,在清除HBV感染同时也造成了肝细胞炎性损伤,其与肝细胞损伤严重程度有一定的相关性.     

慢性乙型肝炎患者病毒载量与IFN-γ、IL-10、CD19~(+)水平的相关性

目的探讨IFN-γ、IL-10、CD19+水平与慢性乙型肝炎患者病毒载量的相关关系。方法收集感染科2012年6月到2014年2月的门诊及住院的慢性乙型肝炎患者80例,同时选取在我院体检正常或者非肝炎病毒感染患者50例作为对照组,荧光定量PCR法定量病毒载量,酶联免疫吸附试验(ELISA)双抗体法检测IFN-γ、IL-10水平及荧光染色测定CD19+水平,分析IFN-γ、IL-10、CD19+水平与HBV DNA定量相关关系。结果病例组的IFN-γ水平低于对照组,而IL-10与CD19+高于对照组中的水平,差异具有统计学意义(P0.05)。HBV DNA定量、IFN-γ、IL-10、CD19+水平在HBV DNA阴性、低拷贝、高拷贝间差别具有统计学意义(P0.05),其中高拷贝与低拷贝组中HBV DNA定量、IL-10及CD19+水平高于HBV DNA阴性组中水平,IFN-γ低于HBV DNA阴性组中的水平,差异具有统计学意义(P0.05),高拷贝HBV DNA定量、IFN-γ、IL-10、CD19+水平与低拷贝间差别具有统计学意义(P0.05)。IFN-γ与HBV DNA定量间呈负相关关系(r=-0.367,P0.05),IL-10和CD19+水平与HBV DNA定量间呈现正相关关系(IL-10 r=0.362、CD19+r=0.348,P0.05)。结论 IFN-γ水平与慢性乙型肝炎患者病毒载量呈现负相关关系、IL-10、CD19+水平与慢性乙型肝炎患者病毒载量间呈现正相关关系,因此IFN-γ、IL-10、CD19+水平可以作为乙型肝炎患者发生炎症特异指标。     

468例CHB患者肝纤维化进展与血清病毒学指标演变关联性探讨

目的探讨慢性乙型病毒性肝炎(CHB)患者肝纤维化进展与血清病毒学标志的演变关系。方法选择468例CHB患者,根据其不同的肝纤维化程度由轻及重(S0-1,S2,S3-4)分为三组,分别统计三组患者肝纤维化分级与HBe Ag状态及HBV DNA载量的关系。结果随着肝纤维化进展(S0-S1→S2→S3-S4),HBe Ag阳性者的比例逐渐下降(60.00%>23.25%>16.75%),HBe Ag阴性的比例逐渐升高(23.53%<33.82%<42.65%),其差异有统计学意义(χ2=35.756,P<0.001);不同血清HBV DNA载量的患者,肝纤维化水平不同(χ2=58.790,P<0.001),随着肝纤维化的进展,血清HBV DNA呈逐渐下降趋势(r=-0.343)。HBe Ag阳性CHB患者中,其肝纤维化进展与HBV DNA载量的差异同样具有统计学意义(χ2=38.011,P<0.001),肝纤维化水平与HBV DNA载量亦呈负相关(r=-0.290)。结论慢性乙型病毒性肝炎患者随着肝纤维化进展(S0-S1→S2→S3-S4),其血清HBe Ag阳性的比例与HBV DNA载量均呈逐渐下降的趋势,其中HBe Ag持续阳性患者的HBV DNA载量随肝纤维化进展表现出相似的特点。     

慢性乙型肝炎患者肝组织中巨噬细胞移动抑制因子的表达及其对炎症活动的影响

目的:检测巨噬细胞移动抑制因子(macrophage migrationinhibitory factor,MIF)在正常肝和慢性乙型肝炎(chronic hepatitis B,CHB)肝组织中表达的差异,探讨MIF的表达与CHB炎症活动及病毒载量的关系。方法:取CHB40例为实验组(HBeAg阳性CHB28例,HBeAg阴性CHB12例),正常肝组织20例为对照组,用免疫组化和原位杂交技术检测两组中MIF和MIFmRNA的表达,并测定患者ALT和AST的水平及HBV DNA载量。结果:MIF和MIF mRNA在实验组中的阳性表达例数明显高于对照组,两组相比差异有显著性意义(P<0.05);实验组肝组织中MIF和MIF mRNA表达程度与ALT和AST呈正相关,1相似文献   

慢性乙型肝炎患者树突状细胞功能与单个核细胞乙型肝炎病毒共价闭合环状DNA的关系

目的 探讨慢性乙型肝炎(CHB)患者外周血单个核细胞(PBMC)及树突状细胞(DC)内HBV共价闭合环状DNA(HBV cccDNA)的存在状况,DC成熟度及功能状态与DC或PBMC中HBV cccDNA载量的关系.方法 分离29例CHB患者和10例健康对照者的PBMC,用重组人粒细胞-巨噬细胞集落刺激因子(GM-CS...     

慢性乙型肝炎患者血清HBV DNA载量与肝组织病理学变化关系研究

探讨慢性乙型肝炎轻度患者血清HBV DNA载量与肝组织病理学变化的关系。方法根据血清HBeAg状态将310例慢性乙型肝炎轻度患者分为HBeAg阳性和HBeAg阴性,应用荧光定量PCR技术检测血清HBV DNA水平,常规进行肝活检术和病理学观察,分析血清HBV DNA载量与肝组织病理学变化的关系。结果224例HBeAg阳性患者肝组织炎症程度以G0（42.0%）为主,纤维化程度以S0（58.0%）为主;86例HBeAg阴性患者炎症程度以G0（29.1%）、G1（30.2%）、G2（34.9%）为主,纤维化程度以S0（51.2%）为主;HBeAg阳性患者血清HBV DNA水平与肝组织炎症程度无明显相关（r=-0.098,P＞0.05）,与纤维化分期间存在负相关关系（r=-0.309,P＜0.01）;HBeAg阴性患者血清HBV DNA水平与肝组织炎症程度存在正相关关系（r=0.306,P＜0.01）,与纤维化程度无明显相关（r=0.112,P＞0.05）。结论 HBeAg阳性与HBeAg阴性患者血清HBV DNA载量与肝组织病理学变化的关系存在显著差别,应区别分析其临床意义。     

慢性乙型肝炎抗病毒疗效与外周血单个核细胞内乙型肝炎病毒DNA水平的关系

目的 探讨慢性乙型肝炎(CHB)抗病毒疗效与达到停药标准时外周血单个核细胞(PBMC)内HBV DNA水平的关系.方法 入选90例经抗病毒治疗达到停药标准的CHB患者,其中应用IFN 44例,应用核苷类药物46例.所有患者均于停药时检测PBMC内HBV DNA,比较阴性组和阳性组治疗前血清HBV DNA水平与达到停药标准时PBMC内HBV DNA的关系,观察停药时PBMC内HBV DNA水平与复发的关系.计量资料采用t检验,计数资料采用X2检验.结果 90例CHB患者停药时,PBMC内HBV DNA阴性组67例,阳性组23例.CHB患者血清HBV DNA阳转率在PBMC内HBV DNA阴性组为13.4%(9/67例),显著低于阳性组的73.9%(17/23例),差异有统计学意义(X2=30.4873,P<0.01).PBMC内HBV DNA阴性组与阳性组在肝病复发ALT升高幅度(t=0.8729,P=0.3913)、停药后复发时间(t=1.9222,P=0.0665)均差异无统计学意义,而在血清HBV DNA反弹幅度则差异有统计学意义(t=2.7493,P=0.0112).5例患者获得HBsAg血清学转换,且均未检测到PBMC内HBV DNA,随访6～12个月无一例复发.PBMC内HBV DNA阳性组治疗前血清HBV DNA水平为(7.2±1.1)lg拷贝/mL,显著高于阴性组的(5.2±2.1)lg拷贝/mL(t=4.3557,P<0.01).结论 经抗病毒治疗达到停药标准的CHB患者,其停药时的PBMC内HBV DNA水平可能是预测抗病毒疗效持久性的重要因素之一.

Abstract：

Objective To explore the relationship between the antiviral effect and peripheral blood mononuclear cell (PBMC) hepatitis B virus (HBV) DNA when the patients reach the standard of withdrawal of antiviral therapy in chronic hepatitis B (CHB).Methods Ninety CHB patients treated with interferon(n=44) or nucleot (s) ide(n=46) who reached the standard of withdrawal of antiviral therapy were recruited.HBV DNA levels in PBMCs were tested at the end of treatment,and its relationship with serum HBV DNA level before treatment in PBMC HBV DNA positive group and negative group were compared.The correlation between HBV DNA in PBMCs at the end of treatment and relapse were explored.Measurement data were analyzed by student t test and enumeration data were analyzed by X2 test.Results Among 90 patients,67(74.4%) were PBMC HBV DNA negative at the end of treatment,and 23(25.6%) were positive.The serum HBV DNA positive conversion rate in PBMC HBV DNA negative patients was 13.4%,which were significantly lower than that in positive group (73.9%) (X2=30. 4873, P<0.01 ). There were no significant differences of alanine aminotransferase (ALT) levels when hepatitis flare (t=0. 8729, P=0. 3913) and relapse time (t=1. 9222, P=0. 0665) between PBMC HBV DNA negative group and positive group after withdrawal of therapy, while the serum HBV DNA rebound was greater in positive group than that in negative group (t=2. 7493, P=0. 0112). There were five patients who achieved hepatitis B surface antigen (HBsAg) seroconversion, whose PBMC HBV DNA were all undetectable, and none relapsed during follow-up for 6-12 months. The pretreatment HBV DNA as level in PBMC HBV DNA positive was (7.2±1.1) lg copy/mL, which was much higher than that in negative group[(5.2±2.1) lg copy/mL] (t=4. 3557, P<0.01). Conclusions In patients who reach the standard of drug withdrawal,PBMC HBV DNA at the end of treatment is an important predictor for durability of antiviral therapy in CHB.     

Kinetics of phytohemaglutinin-induced IFN-γ and TNF-α expression in peripheral blood mononuclear cells from patients with chronic hepatitis B after liver transplantation

AIM: To study the association between host immunity and hepatitis B virus (HBV) recurrence after liver transplantation. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 40 patients with hepatitis B and underwent orthotopic liver transplantation (OLT) before and 2, 4, 8 wk after surgery. After being cultured in vitro for 72 h, the levels of INF-gamma and TNF-alpha in culture supernatants were detected with ELISA. At the same time, the quantities of HBV DNA in serum and PBMCs were measured by real time PCR. RESULTS: The levels of INF-gamma and TNF-alpha in PBMC culture supernatants decreased before and 2, 4 wk after surgery in turns (INF-gamma 155.52+/-72.32 ng/L vs 14.76+/-9.88 ng/L vs 13.22+/-10.35 ng/L, F = 6.946, P = 0.027 < 0.05; TNF-alpha 80.839+/-46.75 ng/L vs 18.59+/-17.29 ng/L vs 9.758+/-7.96 ng/L, F = 22.61, P = 0.0001 < 0.05). The levels of INF-gamma and TNF-alpha were higher in groups with phytohemagglutinin (PHA) than in those without PHA before surgery. However, the difference disappeared following OLT. Furthermore, INF-gamma and TNF-alpha could not be detected in most patients at wk 4 and none at wk 8 after OLT. The HBV detection rate and virus load in PBMC before and 2, 4 wk after surgery were fluctuated (HBV detected rate: 51.4%, 13.3%, 50% respectively; HBV DNA: 3.55+/-0.674 log10 copies/mL vs 3.00+/-0.329 log10 copies/mL vs 4.608+/-1.344 log10 copies/mL, F = 7.582, P = 0.002 < 0.05). HBV DNA in serum was 4.48+/-1.463 log10 copies/mL before surgery and <10(3) copies/mL after OLT except for one with 5.72 x 10(6) copies/mL 4 wk after OLT who was diagnosed as HBV recurrence. The levels of INF-gamma and TNF-alpha were lower in patients with a high HBV load than in those with a low HBV load (HBV DNA detected/undetected in PBMCs: IFN-gamma 138.08+/-72.44 ng/L vs 164.24+/-72.07 ng/L, t = 1.065, P = 0.297 > 0.05, TNF-alpha 80.75+/-47.30 ng/L vs 74.10+/-49.70 ng/L, t = 0.407, P = 0.686 > 0.05; HBV DNA positive/negative: IFN-gamma 136.77+/-70.04 ng/L vs 175.27+/-71.50 ng/L, t = 1.702, P = 0.097 > 0.05; TNF-alpha 75.37+/-43.02 ng/L vs 81.53+/-52.46 ng/L, t = 0.402, P = 0.690 > 0.05). CONCLUSION: The yielding of INF-gamma and TNF-alpha from PBMCs is inhibited significantly by immunosuppressive agents following OLT with HBV load increased, indicating that the impaired immunity of host is associated with HBV recurrence after OLT.     

乙型肝炎后肝硬化失代偿期患者的临床病理分析

目的探讨乙型肝炎后肝硬化失代偿期患者的肝脏组织炎症活动度情况,并分析其与血清HBV DNA、HBeAg、临床生化指标的相关性。方法收集乙型肝炎后肝硬化行脾切除术中取肝脏活检的病例82例,取肝组织行HE、网状纤维和Masson染色,观察肝脏组织炎症活动度。以Spearman秩相关分析临床生化指标与肝脏组织炎症活动度的相关性,并采用行*列表资料的χ2检验比较不同血清HBV DNA载量、HBeAg与肝组织炎症活动度关系。结果高达76.8%(63/82)的乙型肝炎后肝硬化失代偿患者肝脏组织仅有轻度活动性炎症(G0-1、G2级);血清ALB与肝组织炎症活动度呈负相关(r=-0.405,P0.01),PT与肝脏组织炎症活动度呈正相关(r=0.340,P0.01),而血清ALT、TBIL、HBeAg阳性、HBV DNA载量和Child-Pugh评分与乙型肝炎后肝硬化失代偿患者的肝脏组织炎症活动度无相关(P0.05)。结论血清HBV DNA载量并不能反映患者的肝脏组织炎症活动度,乙肝病毒持续复制和肝脏炎性反应坏死是肝炎肝硬化病情进展的主要决定因素。无论肝硬化患者体内病毒复制水平如何,均应尽早进行抗病毒治疗。     

Dynamic changes of HBV DNA in serum and peripheral blood mononuclear cells of chronic hepatitis patients after lamivudine treatment

AIM: To study the dynamic changes of hepatits B virus (HBV) DNA in serum and peripheral blood mononuclear cells (PBMCs) of patients after lamivudine therapy. METHODS: A total of 72 patients with chronic HBV infection were included in this study. All patients were confirmed to have the following conditions: above 16 years of age, elevated serum alanine amonotransferase (ALT), positive hepatitis B e antigen (HBeAg), positive HBV DNA in serum and PBMCs, negative antibodies against HAV, HCV, HDV, HEV. Other possible causes of chronic liver damages, such as drugs, alcohol and autoimmune diseases were excluded. Seventy-two cases were randomly divided into lamivudine treatment group (n=42) and control group (n=30). HBV DNA was detected both in serum and in PBMCs by fluorescence quantitative polymerase chain reaction (PCR), during and after lamivudine treatment. RESULTS: In the treatment group, HBV DNA became negative both in serum and in PBNIC, of 38 and 25 out of 42 cases respectively during the 48 wk oflamivudine treatment, the negative rate was 90.5% and 59.5% respectively. In the control group, the negative rate was 23.3% and 16.7% respectively. It was statistically significant at 12, 24 and 48 wk as compared with the control group (P < 0.005). The average conversion period of HBV DNA was 6 wk (2-8 wk) in serum and 16 wk (8-24 wk) in PBMC. CONCLUSION: Lamivudine has remarkable inhibitory effects on HBV replication both in serum and in PBMCs. The inhibitory effect on HBV DNA in PBMCs is weaker than that in serum.     

Kinetics of phytohemaglutinin-induced IFN-γand TNF-α expression in peripheral blood mononuclear cells from patients with chronic hepatitis B after liver transplantation

AIM: To study the association between host immunity and hepatitis B virus (HBV) recurrence after liver transplantation.METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 40 patients with hepatitis B and underwent orthotopic liver transplantation (OLT) before and 2, 4, 8 wk after surgery. After being cultured in vitro for 72 h, the levels of INF-γ and TNF-α in culture supernatants were detected with ELISA. At the same time, the quantities of HBV DNA in serum and PBMCs were measured by real time PCR.RESULTS: The levels of INF-γ and TNF-α in PBMC culture supernatants decreased before and 2, 4 wk after surgery in turns (INF-γ 155.52±72.32 ng/L vs14.76±9.88 ng/L vs 13.22±10.35 ng/L, F= 6.946, P = 0.027＜0.05; TNF-α80.839±46.75 ng/L vs 18.59±17.29 ng/L vs9.758±7.96 ng/L,F= 22.61, P= 0.0001＜0.05). The levels of INF-γ and TNF-α were higher in groups with phytohemagglutinin (PHA) than in those without PHA before surgery. However,the difference disappeared following OLT. Furthermore,INF-γ and TNF-α could not be detected in most patients at wk 4 and none at wk 8 after OLT. The HBV detection rate and virus load in PBMlC before and 2, 4 wk after surgery were fluctuated (HBV detected rate: 51.4%, 13.3%, 50% respectively; HBV DNA: 3.55±0.674 log(10) copies/mL vs 3.00±0.329 log(10) copies/mL vs 4.608±1.344 log(10) copies/mL, F= 7.582, P= 0.002＜0.05). HBV DNA in serum was 4.48±1.463 log(10) copies/mL before surgery and ＜103 copies/mL after OLT except for one with 5.72×106 copies/mL 4 wk after OLT who was diagnosed as HBV recurrence.The levels of INF-γ, and TNF-α were lower in patients with a high HBV load than in those with a low HBV load (HBV DNA detected/undetected in PBMCs: IFN-γ 138.08±72.44 ng/L vs 164.24±72.07 ng/L, t = 1.065, P = 0.297＞0.05, TNF-α 80.75±47.30 ng/L vs74.10±49.70 ng/L, t= 0.407, P= 0.686＞0.05; HBV DNA positive/negative: IFN-γ 136.77±70.04 ng/L vs 175.27±71.50 ng/L, t= 1.702, P= 0.097＞0.05; TNF-α 75.37±43.02 ng/L vs 81.53±52.46 ng/L, t = 0.402,P = 0.690＞0.05).CONCLUSION: The yielding of INF-γ and TNF-α from PBMCs is inhibited significantly by immunosuppressive agents following OLT with HBV load increased, indicating that the impaired immunity of host is associated with HBV recurrence after OLT.     

慢性乙型肝炎肝组织中程序性细胞死亡因子配体1表达与临床的相关性

目的 探讨程序性细胞死亡因子配体1(PD-L1)在乙型肝炎不同病程期间肝组织中的表达,以阐明其在HBV感染慢性化发生发展中的作用.方法 用免疫组织化学方法结合图像定量分析系统检测65例慢性HBV感染者和5例健康对照者肝组织内PD-L1的表达,并与肝组织炎症活动度分级、TBil、ALT、AST及HBV DNA进行相关分析.结果 健康对照组肝组织PD-L1为(0.64±0.28)%,显著低于慢性乙型肝炎组,分别为:G0(3.07±0.93)%,G1(8.01±1.49)%,G2(11.60±2.60)%,G3(18.41±2.21)%,G4(26.04±3.41)%,其表达量与肝脏炎症活动度分级、TBil、ALT、AST呈正相关(r=0.917、0.787、0.483、0.628,P<0.05),与HBV DNA载量呈负相关(r=-0.620,P<0.05).结论 PD-L1在肝组织中表达上调可能参与了HBV感染慢性化的发病机制.

Abstract：

Objective To investigate expressions of programmed cell death ligand 1 (PD-L1) in hepatic tissues at the different stages of hepatitis B virus ( HBV) infection, and clarify its role in the mechanism of chronic hepatitis B virus infection. Methods The expressions of PD-L1 were detected by immunohistochemistry and computer image quantitative analysis in the hepatic tissues of 65 chronic HBV infected patients and 5 healthy controls. The correlations between PD-L1 expression and inflammatory grading in the hepatic tissues, total bilirubin (TBil), alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum HBV DNA level were analyzed. Results The PD-L1 expressions in hepatic tissues of HBV infection with G0 - G4 inflammatory grades were 3. 07 % ±0.93%, 8.01%±1.49%, 11.60%±2.60%, 18.41%±2.21% and 26. 04% ±3. 41%, respectively,which were all significantly stronger than that in controls (0. 64%±0. 28%). PD-L1 expression was a positively correlated with inflammation grading of hepatitis tissues, TBil, ALT and AST level in serum (r=0. 917, 0. 787, 0. 483, 0. 628; all P<0. 05), and negatively correlated with serum HBV DNA load (r=-0. 620, P<0. 05). Conclusion The upregulated PD-L1 expression may be probably involved in the chronicity of HBV infection.     

乙型肝炎患者外周血单个核细胞中微小RNA的表达变化

目的 探讨乙型肝炎患者外周血单个核细胞(PBMC)中微小RNA(miRNA)和细胞因子的变化及其与炎性反应的相互关系.方法 收集健康对照者17例,急性乙型肝炎发作期、病毒清除期和恢复期患者各20例,慢性乙型肝炎轻度、中度和重度患者各20例和乙型肝炎后肝硬化患者20例.分离PBMC,采用反转录-荧光定量PCR方法检测miRNA146、miRNA155、miRNA181、IFN-α、IFN-β、IFN诱导基因54(ISG54)、IFN调节因子5(IRF5)的表达.多组间比较采用单因素方差分析.结果 急性乙型肝炎患者PBMC中miRNA155的表达水平在急性发作期(2.386±1.835)较高,明显高于健康对照组(1.498±1.276),差异有统计学意义(F=1.137,P=0.045),随疾病进入发作期、病毒清除期(1.633±2.291)、恢复期(0.642±0.836),其表达逐渐降低(F=2.122,P=0.022).同时IFN-α、IFN-β随急性发作期(7.059±9.594、4.767±6.725)、病毒清除期(2.216±2.148、1.750±1.403)和恢复期(0.642±0.836、1.201±0.779)其表达也逐渐降低(F=1.880,P=0.038;F=1.835,P=0.048).相关性分析发现,miRNA155与IFN-α、IFN-β均具有良好的正相关性(r=0.483,P=0.004;r=0.660,P=0.0002).在急性HBV感染患者中,miRNA155的表达与ALT、Tbil均呈正相关(r=0.342,P=0.006;r=0.322,P=0.011),但与血清HBV DNA载量无相关性.miRNA181在HBV感染者PBMC中的表达,除急性乙型肝炎恢复期(1.873±0.998)外,均高于健康对照组(1.307±0.935)(F=2.072,P=0.045),但HBV感染各组间差异无统计学意义.miRNA146的表达水平变化不明显.结论 在HBV感染过程中,miRNAs参与了宿主的抗HBV免疫反应,并与细胞因子表达相关.