氧化苦参碱抑制高糖诱导的大鼠肾小管上皮-间充质转化及其机制研究

 目的：探讨氧化苦参碱(OM)对高糖诱导的大鼠肾小管上皮-间充质转化（EMT）的抑制作用及其可能机制。方法：体外培养大鼠近端肾小管上皮NRK52E细胞,随机分为：对照组、高糖组、高糖+OM不同浓度组和高糖+0.50 g/L OM动态观察组。采用real-time PCR和Western blotting方法检测转化生长因子β1（TGF-β1）、Smad7、α-平滑肌肌动蛋白（α-SMA）、E-钙黏素（E-cadherin）mRNA和蛋白的表达。结果：（1）与对照组相比,高糖组TGF-β1、α-SMA mRNA和蛋白表达水平均进行性增高,Smad7蛋白表达进行性降低,E-cadherin mRNA和蛋白表达进行性降低,呈时间依赖性（P＜0.05）,而Smad7 mRNA表达进行性增高（P＜0.05）;（2）与高糖组相比,高糖+ OM不同浓度组随OM剂量增加,TGF-β1、α-SMA mRNA和蛋白表达均逐渐降低,Smad7蛋白表达水平逐渐增高,E-cadherin mRNA和蛋白表达水平逐渐增高,且呈剂量依赖性（P＜0.05）,而Smad7 mRNA表达无明显差异;（3）与高糖组相比,高糖+0.50 g/L OM动态观察组TGF-β1、α-SMA mRNA和蛋白表达持续降低,Smad7蛋白表达持续增高,E-cadherin mRNA和蛋白表达持续增高（P＜0.05）,而Smad7 mRNA表达无明显差异。结论：OM可抑制高糖诱导的NRK52E细胞发生EMT,其机制可能与OM下调TGF-β1表达及上调Smad7蛋白表达,进而抑制TGF-β1/Smads信号通路的致纤维化效应有关。     

左归丸含药血清通过ERK/TGF-β/Smads信号级联调控MC3T3-E1细胞增殖与分化

目的:研究细胞外信号调节激酶(ERK)/转化生长因子β(TGF-β)/Sma和Mad相关蛋白(Smads)信号级联在左归丸含药血清干预成骨前体细胞系MC3T3-E1细胞增殖与分化中的作用。方法:以倍美力为阳性对照药,对Sprague-Dawley(SD)雌性大鼠灌服高、中、低剂量的左归丸混悬液,7 d后腹主动脉取血分离含药血清。采用噻唑蓝(MTT)法检测左归丸含药血清对MC3T3-E1细胞的增殖作用,采用改良钙钴染色法检测碱性磷酸酶(ALP)表达,采用茜素红染色法检测钙化结节,采用Western blotting法检测核结合因子α1(Cbfα1)和Ⅰ型胶原(ColⅠ)蛋白表达,采用real-time RT-PCR法检测TGF-β₁、Smad4和Smad2 mRNA表达。结果:左归丸含药血清对MC3T3-E1细胞的促增殖作用呈剂量和时间相关性,其中以低剂量且体积分数为15%作用48 h后对MC3T3-E1的促增殖作用最大;左归丸含药血清能促进MC3T3-E1细胞ALP表达,增强细胞基质钙化,提高Cbfα1和ColⅠ蛋白分泌,上调TGF-β₁、Smad4和Smad2 mRNA表达;加入ERK1/2信号通路特异性阻滞剂PD98059后,MC3T3-E1细胞增殖降低,ALP表达下降,细胞基质钙化减弱,Cbfα1和ColⅠ蛋白分泌降低,Smad4和Smad2 mRNA表达下调,TGF-β₁ mRNA表达进一步上调。结论:左归丸可能通过干预ERK/TGF-β/Smads信号级联而调控成骨细胞的增殖和分化,这可能是其防治骨质疏松症的机制之一。     

CAS基因在原发性肝癌组织中的表达及其与HBV感染的关系

目的 探讨细胞凋亡易感基因(CAS)可否作为肝癌的病理诊断标志物,以及肝细胞癌中CAS蛋白的表达与HBV感染之间的关系.方法 应用免疫组化法检测肝癌、癌旁组织及未发生肿瘤的肝硬化、肝炎组织中CAS蛋白的表达情况,同时应用免疫组化法、核酸原位杂交法检测HBV感染的肝细胞癌组织、癌旁组织中HBsAg、HBcAg以及HBV DNA的表达情况,分析肝细胞癌中CAS蛋白的表达与HBV感染之间的关系.结果 CAS蛋白在肝癌组织中的表达较癌旁组织明显升高(P＜0.01),而癌旁组织中CAS蛋白表达较未发生肿瘤的肝硬化、肝炎组织显著增强(P＜0.01).低分化型肿瘤细胞的CAS蛋白表达明显高于中分化型和高分化型(P＜0.01).CAS蛋白在HBV感染的肝细胞癌组织中的表达明显高于非HBV感染的癌组织(P＜0.01),其中HBV DNA阳性的肝细胞癌组织中CAS蛋白表达水平显著高于HBV DNA阴性的肝细胞癌组织(P＜0.05).结论 CAS蛋白在肝细胞癌组织中呈高表达,肝细胞癌分化愈低其表达愈强,表明CAS蛋白可作为肝细胞癌的病理诊断与分化程度的评价标志物.并推测HBV DNA可能通过上调CAS的表达,在HBV感染相关性肝癌的发生发展过程中发挥重要的作用.     

TGF-β1显著上调其信号蛋白Smad2在腹膜间皮细胞中的表达

目的:探讨Smad2信号蛋白在腹膜间皮细胞中的表达及转化生长因子-β1(TGF-β1)对其表达的影响。方法:大鼠腹膜间皮细胞分别培养于不同浓度TGF-β1培养液(0、1.25、2.5、10μg/L)中,用RT-PCR和免疫组化的方法检测不同时间(0、5、15、30、60、120min)Smad2表达的水平和Smad2细胞内定位变化。结果:Smad2mRNA的表达在TGF-β1刺激后5min开始升高,呈时间依赖性,在30min达到高峰,而后逐渐减弱,在120min降至接近正常水平;Smad2mRNA表达也呈浓度依赖性,随TGF-β1浓度的增高而表达增强。磷酸化Smad2蛋白的表达和核内聚集也与mRNA表达一致,呈时间和浓度依赖性。Smad2在TGF-β1刺激下从胞浆转位至核内聚集,并在30min达高峰。结论:腹膜间皮细胞内存在Smad2的表达。TGF-β1可刺激Smad2表达上调和活化,转位至核内发挥作用,并呈浓度依赖和一定的时间依赖性。     

白屈菜红碱对肝纤维化小鼠TGF-β/Smads信号通路的影响

目的:探讨白屈菜红碱对四氯化碳(CCl_4)诱导的肝纤维化损伤小鼠的保护作用及对转化生长因子β(TGF-β)/Smads信号通路的影响。方法:50只C57BL/6N小鼠随机分成正常对照组、模型组及白屈菜红碱低剂量(10 mg·kg~(-1)·d~(-1))、中剂量(20 mg·kg~(-1)·d~(-1))和高剂量(40 mg·kg~(-1)·d~(-1))3个剂量组,每组10只。采用腹腔注射CCl_4和橄榄油混合液8周诱导小鼠肝纤维化模型,白屈菜红碱组于第5周开始灌胃给药。第14周后处死小鼠,观察白屈菜红碱各剂量组干预后小鼠的肝指数,苏木精-伊红染色和苦味酸-酸性品红染色法观察小鼠肝组织的病理改变及肝纤维化的程度;采用分光光度计和酶标仪测定血清中天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、透明质酸(HA)和肝组织中羟脯氨酸(Hyp)的含量;RT-q PCR检测TGF-β1、Smad3、Smad4和Smad7的mRNA表达;Western blot检测TGF-β1、Smad4和Smad7的蛋白表达。结果:与正常对照组比较,模型组肝纤维化的病理改变明显,肝指数、AST、ALT、HA和Hyp均显著升高(P0.05);TGF-β1、Smad3和Smad4的mRNA表达显著上调,Smad7的mRNA表达显著下调(P0.05);TGF-β1和Smad4的蛋白表达显著上调,Smad7的蛋白表达显著下调(P0.05);与模型组比较,白屈菜红碱不同剂量给药组均抑制上述指标的改变(P0.05)。结论:白屈菜红碱能够抑制CCl_4诱导的小鼠肝纤维化,其机制可能与TGF-β/Smads信号通路有关。     

194例慢性乙型肝炎病理及其与血清HBV DNA、HBeAg、ALT关系的研究

目的探讨慢性乙型肝炎病理及其与血清HBV DNA、HBeAg、ALT关系。方法对194例慢乙肝患者进行肝组织病理、HBV免疫组化检查,并检测肝功能、血清HBVM和HBV DNA。结果血清HBeAg阳性组的肝组织G2、G3~4、S2、S3~4发生率与阴性组比较差异有统计学意义,肝组织S0组与S1~4组比较差异有统计学意义,肝组织G0~1组与G2~4组、HBcAg阳性组与阴性组的HBV DNA含量比较差异亦有统计学意义,肝组织HBsAg表达为" "者与" ~ "者血清HBV DNA含量比较差异无统计学意义,肝组织达S1或(和)G2以上者血清ALT水平分别为:<40U/L组占28.57%,40~80U/L组占53.33%,81~400U/L占80.15%,>400U/L组占77.88%。结论血清HBV DNA与肝组织HBcAg表达有一致性,与肝内HBsAg无关,HBV DNA含量低可能是肝组织炎症活动度和纤维化程度高,ASC和轻度肝损害者应争取肝活检,以及时判断肝组织病理程度和治疗时机。     

麦冬抑制大鼠心肌成纤维细胞胶原合成的作用及其机制

 目的：观察麦冬抑制大鼠心肌成纤维细胞胶原合成的作用及其机制。方法：以培养的SD大鼠心肌成纤维细胞为观察对象,随机将心肌成纤维细胞分为4组（n=10）：对照组（未用麦冬处理大鼠心肌成纤维细胞）、麦冬（10 μg/L）组、麦冬（20 μg/L）组和麦冬（30 μg/L）组。测定不同组心肌成纤维细胞活力、［3H］-脯氨酸掺入率、TGF-β1 、p-Smad2/3和Smad2/3 蛋白表达的变化。结果：与对照组比较,麦冬（10　μg/L）组大鼠心肌成纤维细胞活力、［3H］-脯氨酸掺入率、TGF-β1 、p-Smad2/3和Smad2/3表达显著降低（P<0.01）。与麦冬（10　μg/L）组比较,麦冬（20 μg/L）组大鼠心肌成纤维细胞活力、［3H］-脯氨酸掺入率、TGF-β1 、p-Smad2/3和Smad2/3表达显著降低（P<0.01）。与麦冬（20 μg/L）组比较,麦冬（30　μg/L）组大鼠心肌成纤维细胞活力、［3H］-脯氨酸掺入率、TGF-β1 、p-Smad2/3和Smad2/3表达显著降低（P<0.01）。结论：麦冬对大鼠心肌纤维化有明显抑制作用,其机制可能与TGF-β1 、p-Smad2/3和Smad2/3 蛋白表达的降低有关。     

转化生长因子-β和Smad4在绝经过渡期大鼠卵巢颗粒细胞中的表达

目的 探讨转化生长因子-β（TGF-β）、Smad4 在绝经过渡期大鼠卵巢颗粒细胞中的表达,分析其与卵巢功能衰退的关系。 方法 雌性SD大鼠分为对照组（C组,6月龄,阴道涂片筛选,n=9）、绝经过渡期组（MT组,12~14月龄,阴道涂片筛选,n=8）,大鼠麻醉处死后迅速取出卵巢,采用机械分离方法释放卵泡颗粒细胞,于CO₂培养箱中培养,利用免疫细胞化学法检测促卵泡刺激素受体（FSHR）蛋白的表达,鉴定卵巢颗粒细胞;免疫细胞化学法检测TGF-β、Smad4蛋白在两组卵巢颗粒细胞的表达;采用Real-time PCR的方法检测各组颗粒细胞中TGF-β、Smad4 mRNA的表达。 结果 免疫细胞化学显示分离培养的颗粒细胞纯度＞95%,MT组TGF-β、Smad4蛋白表达水平低于对照组（P<0.05）;Real-time PCR结果显示,两组均有TGF-β、Smad4 mRNA的表达,MT组TGF-β、Smad4 mRNA表达水平显著低于对照组（P<0.05）。 结论 TGF-β、Smad4参与绝经过渡期大鼠卵巢功能的衰退过程,绝经过渡期大鼠功能衰退的部分原因可能与卵巢颗粒细胞中TGF-β、Smad4 的表达降低有关。     

转化生长因子-β1与肺癌关系的研究

目的:探讨转化生长因子-β1(TGF-β1)与肺癌的发病关系及放疗对血清TGF-β1水平的影响。方法: 应用免疫组织化学技术检测肺癌组织中的TGF-β1表达;应用酶联免疫吸附法定量测定肺癌患者放疗前、放疗后及随访半年时肺癌患者血清TGF-β1水平,统计分析不同阶段TGF-β1含量变化。 结果: 肺癌组织中TGF-β1表达明显大于对照组;39例肺癌患者放疗前血清TGF-β1水平(11.0±1.5)μg/L高于对照组(3.8±0.2)μg/L(P＜0.05);放疗结束后平均水平为(5.6±0.5)μg/L,明显低于放疗前(P＜0.05),与对照组无明显差异(P＞0.05);随访半年TGF-β1平均水平为(11.3±1.2)μg/L,明显高于放疗结束时(P＜0.05),与放疗前无明显差异(P＞0.05)。各组织类型间及各期病人间TGF-β1水平无明显差异。 结论: TGF-β1与肺癌发病有关,TGF-β1可能是肺癌患者的重要肿瘤标志物。     

自发性高血压大鼠心肌组织microRNA-133a与TGF-β1蛋白表达的改变及意义

 目的：观察microRNA-133a（miR-133a）与转化生长因子-β1（transforming growth factor β1,TGF-β1）蛋白在自发性高血压大鼠（spontaneously hypertensive rats,SHR）心肌组织中的表达改变和关系。方法：取12只18周龄雄性自发性高血压大鼠为SHR组,12只18周龄雄性Wistar-Kyoto （WKY）大鼠为对照组,通过无创血压测量分析系统测大鼠尾动脉血压,Masson染色检测心肌胶原容积分数（collagen volume fraction,CVF）和血管周围胶原面积比率（perivascular collagen area ratio, PVCA）,实时荧光定量PCR检测miR-133a表达水平,免疫组化和Western blotting法检测心肌TGF-β1蛋白表达。结果：与对照组比较,SHR组的收缩压和舒张压明显升高（P<0.01）,心肌CVF和PVCA明显升高（P<0.01）,TGF-β1蛋白表达水平明显升高（P＜0.01）,miR-133a表达水平明显降低（P<0.01）,SHR组心肌miR-133a表达水平为对照组的（23.9±4.6）%;SHR组心肌组织miR-133a与TGF-β1蛋白表达水平呈负相关（r=-0.791, P<0.01）。结论：SHR心肌组织miR-133a表达下调,伴随TGF-β1蛋白表达升高和胶原合成增加。miR-133a与TGF-β1可能参与SHR大鼠的心肌纤维化。     

Core Antigen Expression Is Associated with Hepatic Necroinflammation in e Antigen-Negative Chronic Hepatitis B Patients with Low DNA Loads

Intrahepatic hepatitis B virus (HBV) core antigen (HBcAg) is a hallmark of viral replication in hepatitis B virus e antigen (HBeAg)-positive chronic hepatitis B (CHB). The aim of this study was to evaluate the role of HBcAg in HBeAg-negative CHB. One hundred six HBeAg-negative CHB patients who underwent ultrasonographically guided liver biopsy were reviewed for their HBV DNA load and clinical and histological data. Factors associated with the expression of intrahepatic HBcAg were analyzed. Among the patients, 35 (33%) were positive for HBcAg by immunohistostaining. In patients whose HBV DNA loads were higher than 10⁷ copies (cp)/ml, nearly one-half (52%) had detectable HBcAg. Compared with HBcAg-negative patients, HBcAg-positive patients had higher serum alanine transaminase (ALT) and HBV DNA levels and more-severe hepatic necroinflammation. High serum ALT level (>160 U/liter) and HBV viral load were the determinants of HBcAg expression in multivariate analysis. Large amounts of HBcAg expression were frequently detected in patients with high DNA loads, and the patterns of HBcAg distribution were not related to histological activity or HBV DNA levels. In patients with lower HBV DNA loads, the expression of HBcAg was the key factor associated with active hepatic necroinflammation (hazard ratio = 11.25; 95% confidence interval [CI], 1.42 to 89.26; P = 0.022). In conclusion, the expression of HBcAg is not frequent in HBeAg-negative CHB. The expression of intrahepatic HBcAg indicates active hepatic necroinflammation, even in patients with low HBV DNA load. Both HBV viral load and HBcAg expression have implications in the pathogenesis of HBeAg-negative CHB.Hepatitis B virus (HBV) is a circular, partially double-stranded DNA virus (10, 32). HBV infection often leads to chronic hepatitis when it occurs in the neonatal period or early childhood. The natural history of chronic hepatitis B (CHB) has been divided into 4 phases: immune tolerance, immune clearance, immune control, and reactivation after HBV e antigen (HBeAg) seroconversion (19). HBV c antigen (HBcAg) is an intracellular antigen that is expressed in HBV-infected hepatocytes. HBcAg is an immunogenic protein and plays a role in serving as a target antigen for the host immune reaction. HLA class I- and II-restricted T-cell responses to the HBcAg are required for viral clearance (5). In the HBeAg-positive phase (including immune tolerance and immune clearance phases), HBV DNA levels in serum and liver are high (20, 23) and HBcAg is often present in hepatocytes (7, 14, 26, 30). Generally, after the process of HBeAg seroconversion, the level of HBV DNA declines and the HBcAg is undetectable in hepatocytes (immune control phase) (7). However, the active phase of HBeAg-negative CHB occurs in some patients (reactivation phase) (13, 15). HBeAg-negative CHB is prevalent in Asia and Mediterranean Europe and may lead to cirrhosis and hepatocellular carcinoma (HCC) (15). Mutations in precore (G1896A) and basic core promoter (A1762T and G1764A) regions that stop or decrease the production of HBeAg are the major variants in HBeAg-negative CHB (2, 22, 27, 31, 33). These mutations theoretically do not interfere with the initiation and production of HBcAg. The intrahepatic HBcAg will reappear in some, but not all, HBeAg-negative CHB cases. The clinical significance of HBcAg in HBeAg-negative CHB is unclear.Previous reports have found that the localization and expression level of HBcAg are associated with active liver disease or viral replication in the HBeAg-positive stage (7, 14, 26). In the immune tolerance phase, patients usually have a higher level of HBV viremia and the expression of HBcAg is mainly localized in the nucleus, whereas in the immune clearance phase HBcAg can be expressed in the nucleus, cytoplasm, or both in infected hepatocytes (7, 14, 26). It has been suggested that cytoplasmic expression of HBcAg correlates with the severity of liver damage and that nuclear expression of HBcAg reflects the level of viral replication (8). HBcAg distribution might have a certain correlation with serum aminotransferase, HBV DNA, and HBeAg status (18). However, the study population in previous reports was mainly positive for HBeAg (immune tolerance and immune clearance phases), and only a few patients were in the HBeAg-negative phase. Factors associated with the expression and distribution of intrahepatic HBcAg after HBeAg seroconversion deserve study.Measurement of HBV DNA levels is now a useful test for evaluating HBV replication and is considered a marker to determine not only the start and the endpoint of antiviral treatment but also the risk of HCC development (23, 28). The clinical significance of reappearance of intrahepatic HBcAg in the reactivation phase of CHB based on HBV viral loads had not been well evaluated. In the current study, we tried to clarify the relationship among the localization, degree of intrahepatic HBcAg expression, level of HBV viremia, and pathological findings in HBeAg-negative CHB.     

慢性乙型肝炎患者肝组织中HBV抗原表达特征及其临床意义

目的探讨慢性乙型病毒性肝炎肝活检组织中检测乙肝表面抗原(HBsAg)和乙肝核心抗原(HBcAg)表达强度及表达方式的必要性。方法采用EnVision免疫组织化学法检测196例慢性乙型肝炎患者肝穿组织中HBsAg和HBcAg的表达水平,并用荧光定量PCR检测其血清中的HBV DNA的含量。对肝组织进行炎症活动度分级和纤维化分期。结果肝组织中的HBsAg表达强度和表达方式与炎症分级、纤维化分期和血清乙肝病毒载量均无相关性(P>0.05)。HBcAg表达强度与炎症分级无相关性(r=-0.02,P>0.05);与纤维化分期呈负相关(r=-0.28,P<0.01);与血清乙肝病毒载量呈正相关(r=0.53,P<0.01)。HBcAg表达方式与炎症分级为负相关(r=-0.27,P<0.01),其中浆型组炎症活动度分级高于核型组和混合型组(P<0.01),混合型组高于核型组(P<0.01)。HBcAg表达方式与纤维化分期亦呈较弱的负相关(r=-0.23,P<0.01),其中浆型组纤维化分期高于核型组和混合型组(P<0.05)。HBcAg表达方式与血清乙肝病毒载量呈正相关(r=0.22,P<0.01)。结论区分肝组织中的HBsAg表达强度和表达方式无益于了解慢性乙型肝炎患者肝损害的程度,而检测肝组织中的HBcAg则有助于临床抗病毒治疗。     

慢性HBV感染肝脏病理变化和生化ALT及病毒学关系

目的 探讨慢性HBV感染ALT、HBV DNA与肝脏病理的关系.方法 对81例慢性HBV感染患者检测血清ALT、HBV DNA.并进行肝活检病理检查.结果 肝脏炎症分级和纤维化分期与ALT明显相关(r值分别为0.683和0.419),与HBV DNA无相关性.随着肝脏炎症活动度和纤维化程度的加重,ALT有升高趋势(χ2趋势值分别为25.81和12.012),HBV DNA无升高趋势,而随着HBVDNA的升高,肝脏炎症活动度和纤维化程度并无加重趋势.肝组织HBsAg、HBcAg阳性组与阴性组的ALT、HBV DNA差异无统计学意义.结论 ALT与肝脏炎症活动度有明显相关性,仍是观察炎症变化的敏感指标,HBV DNA与肝组织炎症分级及纤维化分期无相关性.     

肝组织病理及肝内抗原表达与聚乙二醇干扰素α-2a抗乙肝病毒疗效关系的研究

目的观察聚乙二醇干扰素α-2a（PegIFNα-2a）治疗慢性乙型肝炎的疗效,探讨肝脏病理改变和肝细胞内病毒抗原的表达类型与PegIFNα-2a抗病毒疗效的关系。方法选择HBeAg阳性慢性乙型肝炎68例,HBeAg阴性慢性乙型肝炎45例,通过肝组织病理检测,观察肝脏病理改变和肝细胞内病毒抗原的表达类型与PegIFNα-2a治疗后血清HBV DNA的阴转率、HBeAg转换率和完全应答率之间的关系,并随访48周,观察持续应答情况。结果 HBsAg的阴转在不同的炎症活动度间差异无统计学意义;在HBeAg阳性患者中,G1组的HBeAg转换率和完全应答率与G3组比较差异有统计学意义（P均〈0.05）;炎症活动度高的病例经治疗后48周的持续应答率高于炎症活动度低的病例（χ2=4.311,P〈0.05）;肝细胞内HBcAg浆型表达者HBeAg阴转、HBeAg转换率、HBV DNA的阴转率均高于HBcAg核型表达者（P均〈0.05）。结论肝组织病理改变和病毒抗原在肝细胞的表达类型可能成为PegIFNα-2a抗病毒疗效的潜在预测因素。炎症活动度高、肝细胞内HBcAg浆型表达者可能对PegIFNα-2a的治疗应答更好。     

慢乙肝患者外周血中Th17细胞趋化因子受体的表达水平

目的 检测慢性乙型病毒性肝炎（chronic hepatitis B,CHB）患者外周血中Th17细胞表面趋化因子受体CCR4、CCR6、CXCR3的表达,分析CCR4、CCR6、CXCR3与丙氨酸转氨酶（ALT）、总胆红素（TBil）及HBV DNA载量的相关性.方法 流式细胞仪检测30例CHB患者（CHB组）及15名健康人（对照组）外周血中Th17细胞CCR4、CCR6、CXCR3的表达,与ALT、TBil、HBV DNA载量进行相关性分析.结果 CHB组CD4＋ Th17细胞CCR4、CCR6、CXCR3的表达水平高于对照组,其差异有统计学意义（P＜0.05）.CHB组CD8＋ Th17细胞CCR4、CCR6的表达水平高于对照组,其差异有统计学意义（P＜0.05）.CCR4、CCR6表达水平与ALT、HBV DNA载量呈正相关（P＜0.05）,与TBil水平无相关性（P＞0.05）.结论 CHB患者外周血中Th17细胞表面CCR4、CCR6表达增高,与炎症程度相关,可能涉及Th17细胞引起的肝损伤.     

慢性乙型肝炎血清及肝组织病毒学标志与病理损伤的关系

目的探讨慢性乙型肝炎(CHB)患者血清及肝组织病毒学标志与肝组织病理损伤的关系。方法对647例CHB患者血清病毒学标志HBsAg、HBsAb、HBeAg、HBeAb、HBcAb、HBVDNA及其中418例肝细胞病毒学标志HBsAg、HBcAg的表达与肝组织病理损伤进行对比分析。结果CHB患者血清及肝组织病毒学标志与肝组织病理损伤密切相关。结论血清HBsAg、HBeAb、HBcAb阳性,HBVDNA阴性的患者肝组织炎症及纤维化程度较轻;HBVDNA与肝组织炎症分级及纤维化分期无明显相关;肝细胞HBsAg、HBcAg均阴性表达的肝组织炎症及纤维化程度较重。     

Correlation between hepatic hepatitis B core antigen and serum hepatitis B virus-DNA levels in patients with chronic hepatitis B virus infections in Taiwan

Paired liver biopsy specimens and serum samples from 76 patients with chronic hepatitis B virus (HBV) infection were taken for staining of hepatitis B core antigen (HBcAg) by immunoperoxidase and testing of HBV-DNA by a spot hybridization technique, respectively. Thirty-two tissue specimens showed positive staining for HBcAg in their hepatocytes. The two patients with diffuse HBcAg expression in liver tissue also had high serum concentrations of HBV-DNA (greater than 10 pg/10 microL). Among 30 patients with focal HBcAg distribution, 28 patients (93.3%) had measurable levels of serum HBV-DNA and 17 patients (60.7%) had high levels of serum HBV-DNA. Of 44 patients without hepatic HBcAg expression, only 12 patients (27.3%) had detectable serum HBV-DNA, and most patients (93.1% [11/12]) had low concentrations (less than 10 pg/10 microL). Nineteen patients had superimposed hepatitis D virus infection, and, of these, three patients (15.8%) had detectable serum HBV-DNA in low concentrations, while one of the three patients had stainable HBcAg in his hepatocytes with focal distribution. Two of the three patients with hepatitis A virus superinfection who had focal HBcAg expression in their liver tissue had serum HBV-DNA levels that were high during the acute phase of hepatitis A virus infection, and in one patient his serum HBV-DNA levels further increased from 10 pg/10 microL to 40 pg/10 microL during the recovery phase. Thus, measurement of serum HBV-DNA levels in patients with chronic HBV infection correlated well with their hepatic HBcAg expression, and both represent the precise status of HBV replication.     

Flowcytometric quantitation of hepatitis B viral antigens in hepatocytes from regular and fine-needle biopsies

The aim of the study was to investigate the use of flow cytometry, as an alternative for immunohistochemistry, for the detection of viral antigens in the liver of patients with chronic hepatitis B virus (HBV) infection. Hepatocytes were obtained from regular- and fine-needle biopsy from HBV positive (n=17) and negative (n=7) patients and quantified by flow cytometry for intracellular hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg). Number of HBsAg positive hepatocytes ranged from 0 to 83%. A significant correlation was found between the percentage of infected hepatocytes and the intracellular expression level of HBsAg (R=0.841, p<0.001). The specificity and sensitivity of flow cytometry was similar to immunohistochemistry. Of the patients on anti-viral treatment with undetectable serum HBV DNA (<400 copies/ml), two had high HBsAg expression in the liver. HBcAg staining was found in 3 out of 15 patients, with 2-3% positive hepatocytes. The results obtained with fine-needle aspiration biopsy (n=12) were comparable to regular biopsy. In conclusion, flowcytometric quantitation of HBV antigens is sensitive and provides relevant information on the course of infection. The minimally invasive fine-needle biopsy provides a useful alternative for regular-needle biopsy for monitoring intrahepatic antiviral responses during therapy.