乙型肝炎病毒宫内感染研究现状

乙型肝炎(乙肝)病毒(hepatitis B virus,HBV)宫内感染是我国HBV携带者和慢性乙型肝炎(慢乙肝)发生的重要原因。胎儿期的HBV感染不仅易形成以后的慢性携带状态,而且是肝硬化和肝癌的高危因素。对HBV宫内感染的研究主要集中在国     

宫内感染乙型肝炎病毒儿童白细胞介素12转录研究

目的探讨外周血单个核细胞白细胞介素12(IL-12)P40转录变化和宫内感染乙型肝炎病毒(HBV)免疫失败的关系.方法采用体外细胞培养和半定量RT-PCR技术对25例宫内感染免疫失败儿童、8例宫内感染有效儿童及19名正常免疫儿童外周血单个核细胞在丝裂原(植物血凝素和细菌酯多糖)、酵母重组乙型肝炎表面抗原(HBsAg)及无刺激物时IL-12 P40 mRNA转录水平进行检测.结果IL-12 P40 mRNA丝裂原刺激时转录在免疫失败组高于免疫有效儿童(P=0.042),自发转录和血清丙氨酸转氨酶水平呈显著正相关(r=0.389,P=0.004).在小剂量HBsAg刺激时,对照组、有效组和免疫失败组转录差异不明显.和小剂量HBsAg刺激时比较,大剂量HBsAg刺激时对照组表现为转录显著增加(P=0.039),有效组和免疫失败组变化不明显.结论宫内感染HBV儿童对特异性抗原刺激IL-12反应低下可能是导致免疫失败的机制之一,对非特异性刺激反应增强可能与免疫失败儿童的肝细胞损伤有关.     

乙型肝炎病毒宫内感染的传播途径及早期诊断

目的分析羊水、脐带血、母血、胎盘等组织中乙型肝炎病毒标志物(HBV M)及HBV DNA与胎儿感染的关系，探讨HBV母婴传播机制。方法采用微粒子化学发光及核酸扩增杂交梳技术对65例血液乙型肝炎表面抗原(HBsAg)阳性不同孕龄孕妇的羊水、母血、脐带血进行HBV M和HBV DNA检测，对自然流产胎儿或死亡婴儿的胎盘、肝脏、心脏、肺脏进行免疫组织化学检测。结果65例血液HBsAg阳性，不同孕龄孕妇的羊水中HBsAg阳性率为21．50％，脐带血阳性率为20．00％；母血、羊水．脐带血HBsAg、乙型肝炎e抗原(HBeAg)、抗-HBc、HBV DNA均阳性者为6．15％；HBsAg、抗-HBc、抗-HBc阳性、HBV DNA阴性者占13．85％。对4例血液、羊水、脐带血HBsAg、HBeAg、抗-HBc、HBV DNA阳性孕妇分娩或自然流产后胎盘、胎儿及死亡婴儿的肝脏、肺脏、心脏进行免疫组织化学检测发现，胎盘各层组织镜下均可见到HBsAg、HBcAg阳性细胞；在肝脏、肺脏组织中可见到HBsAg、乙型肝炎核心抗原(HBcAg)阳性细胞，心肌组织内未见有HBsAg、HBcAg阳性细胞。结论胎儿感染HBV与羊水、胎盘中的病毒相平行；HBV在宫内可感染胎儿血液、肝脏、肺脏等组织；羊水检测HBV M及HBV DNA可作为胎儿早期HBV感染的诊断依据之一。     

树突状细胞亚群相对数与乙型肝炎病毒复制及肝损伤的关系

目的探讨外周血树突状细胞(DC)亚群相对数量与慢性乙型肝炎(简称慢乙肝)患者血清乙型肝炎病毒(HBV)DNA水平和肝脏病理炎症损伤程度问的关系。方法定量聚合酶链反应法检测患者血清HBV DNA,利用流式细胞仪检测外周血DC亚群。结果血清HBV DNA<106拷贝/ml慢乙肝患者外周血DC2相对数量显著高于HBV DNA≥106拷贝/ml患者和健康者(P<0.05),而后两组间DC2的差异无统计学意义;上述三组中DC1相对数量差异无统计学意义;外周血DC亚群相对数与患者临床型别和肝内炎症损伤程度无关。结论外周血DC2亚群的升高与慢乙肝患者体内HBV低水平复制相关,提示DC2 可能在抑制HBV复制中发挥作用;外周血DC亚群相对数与肝组织炎症程度不相关。     

乙型肝炎病毒宫内传播情况调查

本文调查了275例乙型肝炎病毒血症产妇的静脉血及其所生277名新生儿的脐带血乙型肝炎病毒五项标志物。结果产妇静脉血HBsAg、抗-HBs、HBeAg、抗-HBe和抗-HBc阳性分别为275例(100％),0例(0％)、84例(30.5％)、79例(28.7％)和274例(99.6％);而新生儿脐带血阳性依次分别为41例(14.8％),2例(0.7％),24例(8.7％)、25例(9.0％)和186例(45.5％)。其中84例母血HBeAg阳性者所生85例新生儿脐带血HBsAg、HBeAg和抗-HBc阳性分别为22例(25.9％)、16例(18.8％)和48例(56.5％);较79例母血为抗-HBe阳性者所生79例新生儿脐带血的7例(8.7％)、2例(2.5％)、32例(40.5％)和112例HBeAg、抗-HBe均阴性的产妇所生113例新生儿脐带血的12例(10.6％)、6例(5.4％)、46例(40.7％)明显为高(P<0.01或P<0.05)。提示HBeAg阳性的产妇更易发生宫内传播。     

慢性乙型肝炎病毒携带儿童外周血T细胞亚群变化及其与HBV DNA的关系

乙型肝炎病毒（HBV）感染的发病主要取决于机体的免疫状况。90％HBV感染患儿可成为慢性带病毒状态或慢性乙型肝炎（CHB）患儿,机体免疫功能状况与病毒的长期携带有重要父系．各种免疫细胞特别是细胞亚群之间的调节紊乱,不论原因来自宿主还是源于病毒,都是体内不能清除病毒及任肝内持续复制的主要原因。     

树突状细胞HBsAg疫苗抗乙型肝炎病毒免疫的体外研究

目的 研究人单核细胞来源的树突状细胞（DC）激活的HBsAg特异性细胞毒性T淋巴细胞（CTL）对表达HBsAg的HepG2/S靶细胞的杀伤效应，以探索DC-HBsAg疫苗在抗乙型肝炎病毒（HBV）中的作用。方法 从健康外周血中分离邮单核细胞，在粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素4（IL-4）的作用下培养7d诱导出DC，然后以DC为刺激细胞在体外诱导出HBsAg特异性CTL；将携有HBV-S基因的pLXSN/S重组质粒电击导入肝癌细胞系（HepG2)，建立表达HBsAg的靶细胞模型HepG2/S；用染色法检测HBsAg特异性CTL对HepG2/S靶细胞的杀伤效应。结果 DC激活的HBsAg特异性CTL对HepG2/S靶细胞具有较强的杀伤效应，不同浓度HBsAg(0μg/L，50μg/L和100μg/L）诱导的CTL的杀伤率分别为3.8%,69.5%和85.1%，而CTL对HepG2细胞无明显杀伤效应。结论 由DC激活的HBsAg特异性CTL具有较强制 特异性抗HBV作用。     

慢性HBV感染及其相关肝病患者外周血T细胞亚群的变化

目的观察不同临床类型HBV感染者外周血T淋巴细胞亚群的差异,探讨HBV对人体T细胞免疫的影响及其可能机制,不同类型慢性HBV感染者免疫失衡的规律。方法用流式细胞仪技术检测患者外周血T细胞亚群。慢性HBV感染者318例,其中HBV携带者8例,慢性乙型肝炎231例,肝炎后肝硬化61例,原发性肝癌18例,观察患者的T淋巴细胞亚群、HBV DNA等。同时收集22名健康志愿者的新鲜血检测T淋巴细胞亚群。结果不同临床类型HBV感染者外周血CD3＋T、CD4＋T、CD8＋T细胞百分数低于正常对照组,差别均有统计学意义（P〈0.05或P〈0.01）,CD4＋/CD8＋比正常对照组低,但差异均无统计学意义（P〉0.05）。结论慢性HBV感染随着病情进展,由慢性肝炎→肝硬化→肝癌,直至细胞免疫功能逐渐衰退。慢性HBV感染的不同阶段的细胞免疫紊乱各具特点,就不同类型患者应采用不同的免疫调节治疗手段。     

慢性HBV感染者外周血T淋巴细胞亚群和NK细胞活性变化

探讨慢性HBV感染者外周血T淋巴细胞亚群及NK细胞活性变化情况。应用流式细胞法检测所有研T淋巴细胞亚群和NK细胞。慢性乙肝、肝炎肝硬化和慢性重型乙肝组患者CD3 、CD4 百分率及CD4 ／CD8 比值与正常组比较均有所下降,且慢性重型乙肝组患者CD4 百分率和CD4 ／CD8 比值与正常组比较差异有显著性。各临床类型慢性HBV感染者NK细胞百分率均降低,与正常对照组比较有统计学意义。慢性HBV感染者细胞免疫功能低下。检测T淋巴细胞亚群及NK细胞活性变化对判断病变程度、指导临床治疗具有一定参考价值。     

慢性HBV感染患者T细胞亚群变化规律及可能的意义

目的通过对不同病理阶段的224例HBV感染患者CD4 、CD8 T细胞亚群、HBV DNA等指标的检测结果进行了回顾性分析,以期明确不同病理阶段T细胞亚群变化的规律及可能的临床意义.方法外周血CD4 和CD8 T细胞亚群检测采用流式细胞仪技术;HBV DNA检测采用荧光定量PCR检测;ELISA检测用于血清HBeAg和抗HBe评价.结果肝硬化患者(499.75±304.46,t=-3.12,P=0.041)和肝癌患者(442.10±241.81,t=4.1,P=0.038)CD4 T细胞,显著低于急性肝炎患者(682.21±299.74);肝硬化患者(303.45±225.84,t=3.75,P=0.033)和肝癌患者(296.59±279.51,t=5.39,P=0.026)CD8 T细胞,显著低于急性肝炎患者(542.83±277.35);59例肝硬化患者的CD4 T细胞(477.42±206.39)低于35例HBsAg阳性患者(512.37±261.87),但统计学差异不显著(t=0.76,P=0.62).但前者CD8 T细胞亚群显著高于后者(369.35±203.14 vs 277.81±212.58;t=3.02,P=0.41);肝硬化和肝癌血清HBV DNA阳性患者,均显著低于慢性重型肝炎患者(t=4.21,P=0.038;t=3.92,P=0.041),但肝癌组与肝硬化组间无差异(t=0.49,P=0.37).结论HBV感染肝硬化和肝癌患者CD4 和CD8 T细胞亚群较急性肝炎和健康对照人群显著降低,对于CD8 T细胞亚群显著降低的HBV感染患者,应该排除原发性肝癌的存在.     

T cell immunopathogenesis and immunotherapeutic strategies for chronic hepatitis B virus infection

Hepatitis B is caused by the host immune response and T cells play a major role in the immunopathogenesis. More importantly, T cells not only destroy hepatocytes infected by hepatitis B virus (HBV), but also control HBV replication or eradicate HBV in a noncytolytic manner. Therefore, analysis of T cell immune response during acute and chronic HBV infection is important to develop a strategy for successful viral control, which could lead to immunotherapy for terminating persistent HBV infection. There have been many attempts at immunotherapy for chronic HBV infection, and some have shown promising results. High viral load has been shown to suppress antiviral immune responses and immunoinhibitory signals have been recently elucidated, therefore, viral suppression by nucleos(t)ide analogs, stimulation of antiviral immune response, and suppression of the immunoinhibitory signals must be combined to achieve desirable antiviral effects.     

Hepatitis C virus eradication associated with hepatitis B virus superinfection and development of a hepatitis B virus specific T cell response

BACKGROUND/AIMS: Specific T cell responses during acute hepatitis B and during chronic hepatitis C have been described in detail. However, the T cell responses during the rare setting of acute hepatitis B virus (HBV) infection in the course of chronic hepatitis C that eventually lead to clearance of both viruses are completely unknown. METHODS: We analyzed the virus specific CD4+ and CD8+ T cell response during an acute HBV superinfection in a patient with chronic hepatitis C. RESULTS: The patient eliminated hepatitis C virus (HCV)-RNA and HBV-DNA from serum soon after the clinical onset of acute hepatitis B. The HBV specific T cell response found in this patient corresponds to the typical response that has been described in acute hepatitis B without chronic HCV infection. In contrast the hepatitis C specific immune response was similar to that generally found in chronic hepatitis C despite the fact that the patient also eliminated HCV-RNA. CONCLUSIONS: We hypothesize that the acute HBV infection induced a HBV specific T cell response which was associated with elimination HBV DNA and HCV-RNA, the latter possibly by bystander mechanisms, e.g. via secretion of cytokines. If such a non-specific bystander mechanism which has proven to be effective in the experimental setting and which is formally described here for a single patient can be shown to be a more general phenomenon, it may support the approach with new antiviral strategies, e.g. the induction of non-specific defense mechanisms against HCV.     

脐带血乙肝标志物筛查乙肝病毒宫内感染的探讨

目的探讨脐带血乙肝标志物用于筛查乙肝病毒宫内感染的可能性。方法收集175例HBsAg阳性孕妇新生儿脐带血及24 h外周血,进行乙肝标志物和HBV DNA检测,6月龄时随访查外周血HBsAg。结果脐带血HBsAg阳性的新生儿外周血HBV DNA均为阳性,6月龄随访HBsAg阳性率均为100%。7例出生时注射了HBIG和乙肝疫苗的HBV DNA阳性的新生儿HBsAg随访阴转;8例脐带血HBsAg和HBV DNA均阴性的婴儿因直接母乳喂养HBsAg随访阳转。结论脐带血表面抗原可以作为筛查胎儿乙肝病毒宫内感染的指标。     

胎盘滋养层细胞的乙型肝炎病毒感染与宫内感染机制

目的：检测HBV对胎盘、胎肝和滋养层细胞的感染情况，探讨HBV的宫内感染机制。方法：研究对象包括20例孕妇胎盘组织、6例引产胎儿胎肝组织及体外培养的胎盘滋养层细胞。ELISA法检测孕妇外周血、胎儿脐血和6个月婴儿外周血HBV标志物；荧光定量PCR法检测血清和滋养层细胞中的HBV DNA；免疫组织化学法和免疫荧光法检测胎盘、胎肝组织及滋养层细胞中HBV标志物的表达；末端脱氧核糖核酸转移酶介导的缺口标记法（TUNEL）检测胎盘和滋养层细胞凋亡情况。结果：孕妇血清HBV DNA水平与胎儿脐血HBV DNA水平相关，脐血HBV DNA阳性者其母血HBV DNA〉1.0×10^7拷贝／mL；6例胎盘组织和3例引产胎儿胎肝组织中可见HBsAg免疫组织化学染色阳性细胞，其中1例胎肝组织中发现HBcAg阳性细胞；体外培养滋养层细胞与HBV DNA阳性血清共孵育后，可检测到HBsAg的表达，亦可检测到HBV DNA。体内和体外实验均检测到HBV感染后滋养层细胞凋亡呈增加趋势，且胎盘细胞的凋亡与脐血HBV DNA水平相关。体外实验结果显示，随感染时间的延长，滋养层细胞凋亡呈增加趋势。6个月后，12例新生儿有1例血清HBsAg、HBeAg和抗-HBc阳性，6例抗-HBs阳性。结论：HBV宫内感染的机制可能是通过HBV感染胎盘屏障而使胎儿发生HBV宫内感染。HBV在胎儿组织器官内的定位和复制可能是新生儿发生慢性HBV感染的重要因素。滋养层细胞凋亡可能是胎盘屏障阻断HBV宫内传播的一种保护性机制。     

HBV宫内感染的传播途径及其机理的研究

目的 探讨HBV宫内感染的传播途径及其机理。方法 应用PCR技术检测HBV感染孕妇羊水、阴道分泌物、乳汁、脐血中HBV DNA；免疫组织化学技术检测胎盘组织中HBsAg和HBcAg的表达。结果 HBsAg、HBeAg、抗HBc阳性孕妇的羊水、阴道分泌物、乳汁、脐血中均检测到了HBVDNA，阳性率分别为48．28％（14／29）、27．59％（8／29）、37．93％（11／29）和24．14％（7／29）；健康对照组孕妇的上述样品中均未检出HBVDNA；HBV感染孕妇胎盘组织各层细胞均可表达两种抗原，但阳性细胞数目从母体面到胎儿面逐渐减少，阳性细胞的着色强度逐渐减弱。健康对照组孕妇胎盘组织中未发现HBsAg和HBcAg阳性染色细胞。结论 孕妇感染HBV后可通过多种途径传播，而羊水感染是导致胎儿感染的重要传播途径。     

Selective functional deficit in dendritic cell--T cell interaction is a crucial mechanism in chronic hepatitis B virus infection

Summary. A defect in specific T cell immunity has long been assumed to be the central mechanism of persistent Hepatitis B virus (HBV) infection. Recent studies on HBV transgenic mice have suggested, however, that functional deficit of dendritic cells (DC) was an underlying cause for the T cell dysfunction. The functions of monocyte‐derived DC were determined by studying 75 subjects that included chronic hepatitis B patients with low or high HBV load; antibody to hepatitis B surface antigen (anti‐HBs) positive individuals who had recovered completely from previous acute HBV infection; healthy donors who had received hepatitis B vaccination and were anti‐HBs positive; and immunologically naïve to HBV or the vaccine individual. Impaired interactions between monocyte‐derived DC and T cells were shown in chronic HBV infection patients, especially in those with active virus replication. The dysfunctions included: (i) failure of DC to increase human leukocyte antigen (HLA‐II), B7 expression and interleukin‐12 secretion in responses to hepatitis B surface antigen (HBsAg), (ii) defective induction of T cell proliferative response to HBsAg, (iii) failure to activate T cells to produce cytokines and (iv) deficit in the induction of antigen specific cytotoxic T lymphocytes (CTLs). In vitro treatment of DC with tumour necrosis factor‐α improved HLA‐II and B7 expression, as well as Th cell and CTL responses. It is concluded that defective DC‐T cell interactions may account for the specific T cell immune defects in chronic HBV infection. Immunotherapy that aims at restoring DC functions could offer a new opportunity for effectively managing persistent HBV infections.     

慢性乙肝病毒感染者外周血树突状细胞亚群与血清HBVDNA的关系研究

目的 探讨慢性乙肝病毒感染者外周血树突状细胞(DCs)亚群[髓系DC(MDC)和浆细胞DC(PDC)]的变化与血清HBVDNA的关系.方法 采集健康人和慢性乙肝病毒感染者外周静脉抗凝全血,利用荧光抗体标记和流式细胞仪检测外周血树突状细胞亚群(MDC的特异性标记为Lineage-HLA-DR+CD11c+,PDC的特异性标记为Lineage-HLA-DR+CD123+);固相放免法定量检测血清乙型肝炎病毒标志.结果 慢性乙肝病毒感染者外周血PDC相对平均百分率低于健康对照者,但缺少统计学意义,MDC相对数量在两者间没有差别;血清HBeAg阳性慢性乙型肝炎患者外周血PDC相对数量显著高(P<0.01)于HBeAg阴性患者和健康人(P<0.05),而在上述3组中MDC相对数量无差异(P=0.194).结论 HBV感染可导致患者的免疫功能改变,使机体产生免疫耐受或DC的凋亡;慢性乙型肝炎患者外周血PDC增加与HBeAg血清转换有关,提示PDC可能有抑制病毒的作用.     

隐匿性乙型肝炎病毒感染

大量研究通过对肝组织和血清乙型肝炎病毒（HBV）DNA或转录体的检测，证实隐匿性HBV感染是所谓“隐源性肝炎”及其它慢性肝病的常见病因。现就其发生率、形成机制、临床意义、诊断、治疗等方面作一综述。     

MiR-122 in hepatitis B virus and hepatitis C virus dual infection

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the most common causes of chronic liver diseases and hepatocelluar carcinomas. Over the past few years, the liver-enriched microRNA-122 (miR-122) has been shown to differentially regulate viral replication of HBV and HCV. It is notable that the level of miR-122 is positively and negatively regulated by HCV and HBV, respectively. Consistent with the well-documented phenomenon that miR-122 promotes HCV accumulation, inhibition of miR-122 has been shown as an effective therapy for the treatment of HCV infection in both chimpanzees and humans. On the other hand, miR-122 is also known to block HBV replication, and HBV has recently been shown to inhibit miR-122 expression; such a reciprocal inhibition between miR-122 and HBV suggests an intriguing possibility that miR-122 replacement may represent a potential therapy for treatment of HBV infection. As HBV and HCV have shared transmission routes, dual infection is not an uncommon scenario, which is associated with more advanced liver disease than either HBV or HCV mono-infection. Thus, there is a clear need to further understand the interaction between HBV and HCV and to delineate the role of miR-122 in HBV/HCV dual infection in order to devise effective therapy. This review summarizes the current understanding of HBV/HCV dual infection, focusing on the pathobiological role and therapeutic potential of miR-122.