慢性乙型肝炎肝组织中T-bet的表达及临床病理意义

目的探讨慢性乙型肝炎(chronic hepatitis B, CHB)患者肝组织中T-bet的表达,并分析其与机体免疫和病理发展变化的关系。方法采用免疫组化法检测62例CHB组织中T-bet、CD4的表达,并分析两者表达的相关性。结果在CHB组织中,T-bet蛋白主要在汇管区内表达。肝汇管区T-bet表达与CHB炎症活动度呈正相关(r=0.869,P0.01),与CHB纤维化程度呈正相关(r=0.423,P0.01),与CD4细胞数呈正相关(r=0.938,P0.01)。结论 T-bet表达与CHB的发生、发展关系密切,可能在CHB机体免疫应答中发挥着重要作用。     

慢性乙型肝炎肝组织FasL的免疫组化研究

目的通过研究程序性细胞死亡相关分子ＦａｓＬ在肝组织的表达情况，探讨慢性乙肝发病机制。方法以免疫组化方法检测６８例慢性乙肝活检组织。结果ＦａｓＬ主要表达在浸润的单个核细胞浆、肝细胞浆，其表达程度随组织炎症坏死程度加重而增加。结论ＣＴＬ通过Ｆａｓ机制导致感染的肝细胞凋亡是慢性乙型肝炎发病及炎症活动的重要机制之一。ＦａｓＬ阳性肝细胞可能发挥细胞毒效应，淋巴细胞凋亡的“顺序”和“转式”作用机制有可能存在于慢性肝炎的肝细胞。     

HSP70在慢性乙型肝炎患者肝组织中的表达及意义

目的探讨热休克蛋白 70 ( HSP70 )在慢性乙型肝炎肝组织中的表达及意义。方法采用免疫组化技术对 3 6例慢性乙型肝炎和 2 0例正常肝组织中 HSP70的表达进行检测。结果慢性乙型肝炎和正常肝组织中肝细胞 HSP70表达阳性率分别为 4 5 %和 15 % ,两者相比差异显著 ( χ2 =6.3 ,P<0 .0 5 ) ;中度和重度肝炎 HSP70阳性率明显高于轻度肝炎 (阳性率分别为 62 .5 %和 3 0 % ,χ2 =3 .9,P<0 .0 5 ) ;HSP70阳性细胞多位于灶性和碎屑样坏死区。结论肝细胞 HSP70的异常表达在慢性乙型肝炎免疫保护中起重要作用 ,可作为肝组织损伤的一种标志。     

HBx诱导小鼠肝前体细胞上皮-间质转化

目的 探讨乙型肝炎病毒x蛋白(HBx)能否诱导肝前体细胞(HPCs)发生上皮-间质转化(EMT).方法 将重组质粒pSEB-Flag-HBX与包装质粒pAmpho共同转染人胚胎肾上皮细胞系293T细胞,包装获得携带HBx基因的重组逆转录病毒,并感染小鼠肝前体细胞HP14.5,经稻瘟菌素(blasticidin)筛选,获得稳定表达HBx的HP14.5细胞.观察其形态变化,通过实时定量PCR(real time-PCR)及Western blot法检测其间质细胞标志物和上皮标志物在mRNA和蛋白水平的表达量,并采用划痕实验检测其细胞迁移能力的变化.结果 HP14.5细胞稳定表达HBx蛋白后细胞由多角形变为长梭形;与对照组相比,神经钙黏素(N-cadherin)、Snail、vimentin的mRNA和蛋白水平表达均升高,具有统计学差异(P＜0.05)而上皮性钙黏素(E-cadherin)、细胞角蛋白18(CK18)的mRNA和蛋白表达量显著降低(P＜0.05;同时其迁移能力显著升高(P＜0.05).结论 HBx能诱导肝前体细胞发生EMT,并增强其迁移能力.     

肝细胞癌和癌周肝组织免疫组化研究

本文对２６例肝癌及癌周肝组织进行甲胎蛋白，抗胰蛋白酶，乙型肝炎表面抗原，核心抗原，抗－ＧＢｃ，纤维连接蛋白，溶菌酶，组织细胞抗原，Ｓ－１００蛋白免疫酶标记观察。结果发现在肝癌细胞及癌周非癌肝细胞都能检出表达ＨＢｓＡｇ和ＨＢｃＡｇ的阳性细胞，提示肝癌细胞和癌周肝细胞的ＨＢｓＡｇ和ＨＢｃＡｇ的表达形式相同，推测ＨＢＶ感染和肝癌发病有直接或间接作用。本组研究尚提示ＨＢｃＡｇ和肝癌发病也有关，肝癌细胞分化     

正常成年大鼠神经元前体细胞的迁移

目的探讨正常成年大鼠脑中神经元前体细胞的迁移路径和迁移特征,为病理状况下的神经元迁移研究提供理论和实验基础。方法将成年大鼠脑组织按冠状位和矢状位行冰冻切片（片厚20μm）,采用免疫组织化学染色技术观察Doublecortin（DCX）免疫阳性细胞的迁移路径以及DCX阳性细胞的迁移特征。结果正常成年大鼠神经元前体细胞的迁移主要有两条路径：一条是从室下区到嗅球的吻侧迁移流,另一条是位于皮层和胼胝体之间的胼胝体周围迁移流。吻侧迁移流中的DCX免疫阳性细胞较规则的有序排列,形态上呈梭形的胞体和单个较长的前导突起,前导突起基本朝向嗅球,细胞大小基本一致：而胼胝体周围迁移流中的DCX免疫阳性细胞胞体呈类圆形,突起的大小、数目和方向多样。结论研究神经元前体细胞的迁移不仅有助于明确其迁移的具体机制,而且有助于控制神经元的迁移和设计有效的治疗策略。     

慢性乙型肝炎肝内HBV DNA和HBsAg的双标记定位研究

应用原位杂交和免疫组化PAP法双标记技术,结合病人乙型肝炎(乙肝)病毒血清学标志物检测结果,研究了31例慢性乙肝病人肝穿刺组织中乙型肝炎病毒DNA(HBV DNA)和HBsAg的分布及意义。结果显示,肝组织内检出HBV DNA 23例,HBsAg 26例,二者同时检出者21例。从同组病人肝组织内HBV DNA和HBsAg双标检测结果与其乙肝病毒血清学标志物检测结果的比较来看,肝组织内HBV DNA和HBsAg同时阳性很可能表明HBV正处于复制及表达的活动状态。     

稳定表达HBx的肝前体细胞株的构建及其对增殖的影响

目的 构建稳定表达乙型肝炎病毒X蛋白(HBx)的肝前体细胞株,检测对肝前体细胞增殖,细胞周期及Wnt/β-catemn信号通路的影响.方法 重组质粒pSEB-Flag-HBx与包装质粒pAmpho共同转染人胚胎肾上皮细胞系293T细胞,包装获得携带HBx基因的重组逆转录病毒,并感染小鼠肝前体细胞HP14.5,稻瘟菌素(blasticidin)筛选出稳定表达HBx基因的细胞克隆并扩大培养(HP14.5/HBx组),设HP14.5/Rv(空质粒pSEEB-Flag感染组)及空细胞组HP14.5组为对照组.MTS比色法检测各组细胞的增殖活力,流式细胞术检测细胞周期中各时相所占的比例,荧光定量PCR检测cyclin D1和c-myc mRNA的表达量,Western blot法检测HBx、GSK3β、p-GSK3β(ser-9)、β-catenin、cyclin D1以及c-myc等蛋白的表达.结果 RT-PCR和Western blot法检测到HBx基因和蛋白阳性表达,与对照组相比,HP14.5/HBx细胞增殖活力显著增加(P＜0.05);G1细胞比例明显减少(P＜0.05),S期和G2细胞比例显著升高(P＜0.05);cyclin D1和c-myc mRNA的表达量显著升高(P ＜0.05); GSK3β蛋白水平表达无明显变化(P＞0.05),p-GSK3β、β-catenin、cyclin D1和c-myc蛋白的表达水平显著升高(P＜0.05).结论 成功筛选出稳定表达HBx的肝前体细胞株,HBx通过活化Wnt/β-catenin信号途径促进肝前体细胞的增殖.     

复方木鸡冲剂治疗慢性乙型肝炎的回顾性研究

目的:观察复方木鸡冲剂在慢性乙型肝炎中的疗效。方法:选择28例服用复方木鸡冲剂及护肝药物的慢性乙型肝炎患者作为治疗组,31例仅用护肝药物的患者作为对照组,观察并比较两组治疗前、治疗结束时、治疗结束3月后临床症状改善率、肝功能、血清肝纤维化指标、部分免疫学指标、血常规及超声诊断参数的变化。结果:复方木鸡冲剂可明显改善患者多项检测参数,包括ALT、AST、r-GT、HA、LN、PCⅢ、IgG、r-球蛋白、AFP、血小板、脾厚度和肝光点异常率,部分作用较为持久。结论:复方木鸡冲剂具有保护肝细胞、抑制肝纤维化、降低AFP水平和减轻脾肿大的作用,对慢性乙肝有良好疗效。     

Expression of deleted in malignant brain tumours 1 (DMBT1) relates to the proliferation and malignant transformation of hepatic progenitor cells in hepatitis B virus-related liver diseases

Deng H, Gao Y‐B, Wang H‐F, Jin X‐L & Xiao J‐C
(2012) Histopathology  60, 249–260
Expression of deleted in malignant brain tumours 1 (DMBT1) relates to the proliferation and malignant transformation of hepatic progenitor cells in hepatitis B virus‐related liver diseases Aims: The molecular mechanisms underlying proliferation and malignant transformation of hepatic progenitor cells (HPCs) remain largely unknown. The purpose of this study was to evaluate the correlation between the expression of deleted in malignant brain tumours 1 (DMBT1) and the biological behaviour of HPCs in different hepatitis B virus (HBV)‐related human liver diseases. Methods and results: Expression of DMBT1 in HPCs was investigated by double immunofluorescence labelling in control‐group and HBV‐related liver diseases, including hepatitis, hepatocellular carcinoma (HCC), non‐tumoral liver tissue away from HCC, non‐tumoral cirrhotic tissue adjacent to HCC, and non‐HCC cirrhosis. DMBT1‐positive HPCs were isolated by laser capture microdissection and subjected to duplex polymerase chain reaction in order to detect homozygous deletion of DMBT1. The number of DMBT1‐positive HPCs increased in direct proportion to inflammation severity. Loss of heterozygosity for DMBT1 was more frequent in HCC tumour area and non‐tumoral cirrhotic tissue adjacent to HCC, compared with other HBV‐related liver diseases (P < 0.05). Conclusions: DMBT1 may play an important role in the proliferation of HPCs in HBV‐related liver diseases. Moreover, down‐expression of DMBT1 might enhance the risk of malignant transformation of HPCs.     

慢性乙型肝炎患者外周血单个核细胞Toll样受体3的表达降低

目的 探讨慢性乙型肝炎患者外周血单个核细胞Toll样受体3(TLR3)的表达及其临床意义.方法 分别采集慢性乙型肝炎患者和健康志愿者外周血,荧光定量PCR法检测血清HBV DNA复制水平;使用RT-PCR、流式细胞术以及免疫印迹技术分别检测外周血单个核细胞TLR3的mRNA、蛋白的表达;使用ELISA法检测血清中肿瘤坏死因子α(TNF-α)和干扰素β(IFN-p)水平.结果 慢性乙型肝炎患者外周血单个核细胞中的TLR3表达显著低于健康志愿者,且降低水平与血清HBV DNA复制水平相关;慢性乙型肝炎患者外周血TNF-α、IFN-β浓度显著低于健康志愿者,且降低的水平与血清HBV DNA复制水平相关.结论 慢性乙型肝炎患者外周血单个核细胞TLR3的表达与乙肝病毒的复制水平相关.     

流式细胞术检测慢性乙型肝炎患者外周血Th17和TC17细胞

目的建立流式细胞术检测外周血中Th17、TC17细胞的方法,并探讨慢性乙型肝炎(CHB)患者外周血Th17细胞、TC17细胞变化的的意义。方法用三色流式细胞术检测了24例CHB患者和15例健康对照者外周血中Th17细胞和TC17细胞。结果用流式细胞仪结合不同荧光素标记的CD3、CD8和IL-17A单克隆抗体可准确检测外周血中Th17、TC17细胞的数量。对照组中Th17、TC17的检测结果分别为(2.13±0.50)%(1.6%～3.3%)和(0.27±0.13)%(0.2%～0.6%);CHB患者组中分别为(5.58±4.52)%(1.6%～19.0%)和(1.05±0.93)%(0.2%～3.1%)。CHB患者外周血中Th17、TC17细胞显著高于对照组(P=0.007、P=0.004)。结论流式细胞术检测外周血Th17、TC17细胞方法可靠;CHB患者外周血中Th17细胞、TC17细胞与对照组比较显著增高。     

SIRT3与慢性乙型肝炎肝细胞脂肪变性的关系

目的:分析慢性乙型肝炎(chronic hepatitis B,CHB)合并肝细胞脂肪变性的病理学表现,探讨SIRT3与肝组织炎症反应及脂肪变性的关系.方法:回顾性分析78例CHB肝穿刺活检标本的病理学资料,用免疫组织化学法检测SIRT3在肝组织标本上的表达.结果:78例CHB肝细胞脂肪变性的发生率为33.33%,CHB的炎症程度与肝细胞脂肪变性的发生差异无统计学意义(P>0.05).CHB合并脂肪变以肝细胞轻度脂肪变为主,脂肪变性的病理类型以大泡性脂肪变性多见.SIRT3在非脂肪变性的肝细胞主要定位在细胞质,在脂肪变性的肝细胞定位在脂滴周围的细胞质.本研究发现在非脂肪变性的CHB中,轻度CHB的SIRT3表达要高于中度及重度组,差异有统计学意义(P<0.05),而中度及重度组SIRT3的表达差异无统计学意义(P>0.05).在脂肪变性的CHB,SIRT3的表达与脂肪变性程度的差异无统计学意义(P>0.05).结论:CHB合并肝细胞脂肪变性并不少见,SIRT3在非脂变CHB肝细胞的表达随炎症程度轻度组要高于中度及重度组,而在CHB合并肝细胞脂肪变性组未发现其对脂变程度有何直接联系,但其能很好的勾勒出脂变的大小和形状对我们判断肝细胞脂变的分型有一定的帮助.     

Changes in hepatitis B virus DNA-polymerase activity in patients with chronic infection

Levels of serum hepatitis B virus DNA-polymerase (HBV-DNAP) were studied longitudinally over variable periods of time in 16 HBV chronic carriers using a modified assay procedure developed to increase reproducibility. Ten patients were tested on a short-term basis at 3- to 6-hr intervals for 48 hr or at 24- to 48-hr intervals for 15 consecutive days, and all showed marked vacillations in enzyme levels although hospitalized and untreated. Patients with chronic active hepatitis on liver biopsy had larger fluctuations compared to cases of chronic persistent hepatitis. Six patients were longitudinally tested over a period of 12–32 months and those three receiving immunosuppressive drugs showed a progressive increase in DNAP levels during therapy, but two returned to pretreatment levels after therapy was withdrawn and one even cleared permanently the complete virus with seroconversion to anti-HBe. Such outcome was also observed in one patient with chronic active hepatitis who remained untreated.     

Peroxidase-anti-peroxidase detection of hepatitis B surface and core antigen in liver biopsy specimens from patients with chronic type B hepatitis

Liver biopsy specimens from 58 American patients with chronic type B hepatitis were investigated for the presence and distribution of the hepatitis B core (HBcAg) and surface (HBsAg) antigens by peroxidase-anti-peroxidase techniques. HBsAg was detected in 43 (77%) and HBcAg in 52 (90%) patients. HBcAg was present in 50 of 51 (98%) patients with hepatitis B e antigen (HBeAg) but in only two of seven (29%) of patients with antibody to HBeAg (anti-HBe). There was no correlation between severity of hepatitis or height of aminotransferase activities and the amount of HBsAg or HBcAg in hepatocytes but there was a positive correlation between amount of HBcAg and height of HBV-DNA and DNA polymerase activity in serum. Follow-up liver biopsies, taken 1 to 3 yr later, were available from 39 patients. HBcAg remained detectable in 25 of 26 patients with persistence of HBeAg but disappeared in 12 patients who had lost HBeAg. In nine patients, HBcAg was cytoplasmic as well as nuclear in distribution. Seven of these patients had an intense lobular hepatitis with marked elevations in aminotransferase activities. These findings indicate that the amount of HBcAg in liver correlates with the amount of serum hepatitis B virus as quantified by serum levels of DNA polymerase and HBV-DNA. The amount of nuclear HBcAg does not correlate with the severity of the liver disease, but the presence of cytoplasmic HBcAg usually reflects an active and severe ongoing hepatitis.     

慢性丙型肝炎发生肝脂肪变与血清瘦素的关系

目的 探讨慢性丙型肝炎(chronic hepatitis C,CHC)患者发生肝脂肪变与血清瘦素水平的关系.方法 将未治疗的成年CHC患者80例分为有肝脂肪变组(8例)与无肝脂肪变组(72例),以单纯非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)26例作为对照组,比较各组血清瘦素等生化指标有无差异.结果 CHC有肝脂肪变组ALT、AST水平高于NAFLD组(t=2.10,P＜0.05;t=3.56,P＜0.05),血清总胆固醇、低密度脂蛋白胆固醇、载脂蛋白A和B水平低于NAFLD组(t=-2.78,P＜0.05;t=-3.23,P＜0.05;t=-3.81,P＜0.05;t=-2.76,P＜0.05),两组瘦素水平差异无统计学意义(t=-0.30,P＞0.05).两组CHC的ALT、AST、FPG、TG、TC、LDL-C、APO-A、APO-B、胰岛素抵抗指数、HCVRNA水平、血清瘦素水平和体质量指数差异均无统计学意义(t值分别为-1.25,0.29,0.16,-0.53,0.90,1.58,1.93、0.40、0.24,-0.84,-0.05、-0.91,P均大于0.05).血清瘦素水平女性显著高于男性(t=2.22,P＜0.05).结论 CHC发生肝脂肪变时炎症程度重于NAFLD,胆固醇代谢异常程度轻于NAFLD.血清瘦素水平女性高于男性.CHC发生肝脂肪变与血清瘦素水平无关.     

Predictors of hepatic steatosis in HBeAg-negative chronic hepatitis B patients and their diagnostic values in hepatic fibrosis

Objective: To investigate predictors of hepatic steatosis in HBeAg-negative chronic hepatitis B (CHB) patients and their diagnostic values in hepatic inflammation and fibrosis. Methods: A total of 106 HBeAg-negative CHB patients with clinically and pathologically proven steatosis and 98 patients without steatosis were recruited into this study. The levels of fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG), cholesterol (CHOL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (Alb), globulin (Glb), HBV DNA, body mass index (BMI), homeostatic model assessment of insulin resistance (HOMA-IR) and pathological changes of the liver in inflammation, fibrosis and fatty deposition were examined in all patients. Results: The levels of BMI, HOMA-IR, FBG, insulin, TG, and CHOL were significantly higher in patients with steatosis than those without steatosis (all P<0.05). But ALT, AST and HBV DNA levels were significantly lower in patients with steatosis (all P<0.05). Logistic regression analysis showed that only FINS was a significant predictor for hepatic steatosis (P<0.05); FINS and Glb were significant predictors for hepatic inflammation (all P<0.05); BMI and TC were significant predictors for hepatic fibrosis (all P<0.05). Conclusions: Hepatic steatosis, a common disease in HBeAg-negative CHB patients, was positively associated with BMI, FBG, FINS, TG, TC, GGT, ALP and HOMA-IR. In these patients, the prevalence of hepatic inflammation and fibrosis was also increased.