基质金属蛋白酶组织抑制因子mRNA及蛋白在肝硬化组织中的定位

目的：了解基质金属蛋白酶组织抑制因子TIMP－1和TIMP－2蛋白及mRNA在肝硬化组织中的表达和分布状态。方法：以抗TIMP－1和TIMP－2单克隆抗体及TIMP－1和TIMP－2 cDNA探针为试剂,采用免疫组织化学法和原位杂交技术检测肝硬化组织中TIMP－1和TIMP－2蛋白和mRNA,结果：40例肝硬化患者肝组织中TIMP－1和TIMP－2蛋白以及mRNA表达的阳性率为100％,TIMP－1阳性的组织TIMP－2亦为阳性,且TIMP－1强度高于TIMP－2,而正常肝组织未见阳性表达,TIMP－1和TIMP－2蛋白及mRNA表达的阳性信号均位于肝细胞胞浆中,细胞核中无表达,结论：肝硬化患者的肝细胞中存在TIMP－1和TIMP－2蛋白及mRNA的表达,其表达强度与肝组织病理改变呈正相关,这可能是通过抑制基质金属蛋白酶（MMPs)的活性,使得细胞外基质（ECM）降解减少而引起肝硬化。     

转化生长因子-1和基质金属蛋白酶抑制因子-1在肝硬化组织中的表达及其临床意义

目的了解转化生长因子-1（TGFβ1）和基质金属蛋白酶组织抑制因子-1（TIMP1）在肝硬化组织中的表达和分布状态。方法采用免疫组化法（S—P）。检测10例对照肝组织,57例肝硬化患者肝组织中TGFβ1、TIMP1的表达。结果57例肝硬化患者肝组织中TGFβ1、TIMP1蛋白表达的阳性率分别为90．38％、100％,而正常肝组织未见阳性表达,差异有统计学意义（P〈0．01）。TGFβ1、TIMP1表达的阳性信号均位于肝细胞胞浆中,细胞核中无表达。结论（1）肝硬化患者肝细胞中存在TGFβ1、TIMP1的表达,其表达强度与肝组织病理改变相关。提示TGFβ1与TIMP1的表达与肝纤维化活动状态密切相关,在肝纤维化的形成和发展中具有重要意义。（2）检测TGFβ1、TIMP1的表达可对肝硬化病变从分子水平进行评估,也可作为早期诊断及临床治疗和判断预后的分子病理学指标。     

肝硬化患者肝组织中TIMP-1、TIMP-2的表达

目的 了解TIMP-1和TIMP-2在肝硬化患者肝组织中的表达状态,探讨TIMP-1和TIMP-2在肝硬化发病机理中的作用。方法 以抗TIMP-1和TIMP-2单克隆抗体(McAb)为试剂,采用免疫组织化学法检测肝硬化患者肝组织中TIMP-1和TIMP-2相关抗原的表达。结果 30例肝硬化病人肝组织中TIMP-1和TIMP-2相关抗原表达的阳性率为100％,TIMP-1阳性的肝组织中TIMP-2亦为阳性,且TIMP-1强度高于TIMP-2,而正常肝组织无一例表达阳性。TIMP-1和TIMP-2抗原表达阳性信号主要位于肝细胞胞浆中,未见细胞核表达。结论 肝硬化患者肝细胞中存在TIMP-1和TIMP-2相关抗原表达,TIMP-1和TIMP-2与肝病的进展相关,它通过抑制MMPs的活性,使得ECM降解减少而导致肝纤维化,以致肝硬化。     

肝炎患者肝组织中庚型肝炎病毒相关抗原的表达

目的 研究庚型肝炎病毒（ＨＧＶ）相关抗原在肝组织中定位。方法 采用免疫组织化学方法,用抗－ＨＧＶ非结构蛋白５（ＮＳ５）单克隆抗体（ＭｃＡｂ）对１９６例肝炎患者肝组织中的ＨＧＶ相关抗原进行了检测。结果 １９６例肝炎患者肝组织中的ＨＧＶ相关抗原表达阳必国２５．０％;在血清中ＨＧＶＲＮＡ呈阳性患者肝组织中抗原检出率为３８．３％;单一感染者肝组织中抗原表达阳性率为６６．７％;阳性信号位于胞浆中,阳性信号表     

原发性肝癌雌二醇及其受体的表达及意义

目的通过检测肝癌组织雌二醇（E2）及其受体（ER）表达，探讨雌二醇及其受体与肝癌的关系和意义。方法应用组织芯片和免疫组化S-P法检测48例肝癌、30例乙型肝炎肝硬化组织E2和ER表达．以正常肝组织为对照组，分析E2和ER与肝癌的关系和意义。结果肝癌组、肝硬化组及对照组E2阳性率分别为62．5％、56．7％、10．0％，肝癌组和肝硬化组阳性率均较对照组明显增加（P〈0．01）．肝癌组与肝硬化组两者无显著性差异；肝癌组ER阳性率（43．8％）与肝硬化组ER阳性率（63．3％）相比无显著性差异，但均显著高于对照组（15．0％，P〈0．05）。结论肝癌及肝硬化存在E2、ER高表达。在肝硬化期就已存在E2、ER含量增多，在肝硬化向肝癌的转变过程中，E2、ER发挥重要的促进作用。E2、ER与肝癌的发生发展有密切关系。     

固相致敏红细胞吸附技术快速检测肝硬化患者血清中TIMP-1

目的 建立一种新的固相致敏红细胞吸附技术(SPASE)用于快速检测肝硬化患者血清中TIMP-1。方法 用TIMP-1单克隆抗体包被微量血凝板,加热冲洗后加入待检血清标本,最后加入单克隆抗体致敏红细胞。应用致敏红细胞作为指示系统,观察红细胞吸附状况来判定结果。结果 SPASE法全过程仅需要2.5～3小时,408例肝病患者血清检测TIMP-1结果为急性肝炎组检出阳性率16.41％;慢性肝炎组阳性率为33.31％;肝硬化组阳性率为73.58％。结论 血清中TIMP-1的变化可作为肝纤维化较为有用的诊断指标。SPASE法具有较高的灵敏性、特异性和快速性,试剂用量少,且不需复杂的仪器设备,加之实验程序简单,更适于医院及基层医疗单位应用。     

乙型肝炎患者肝组织中HBV和Fas抗原表达

目的：探讨乙型病毒性肝炎（ＨＢＶ）患者组织中病毒抗原与Ｆａｓ抗原表达的关系。方法：采用免疫组化双标记技术，以鼠抗－ＨＢｓ单克隆抗体，兔抗－ＨＢｃ及兔抗－Ｆａｓ多克隆抗体，对急性普通型，慢性活动型肝炎及活动性肝硬化各２０例肝组织中ＨＢＶＡｇ及Ｆａｓ抗原的表达及分布进行了检测和比较，结果：６０例肝组织中ＨＢｓＡｇ，ＨＢｃＡｇ和Ｆａｓ抗原的检出率分别为８１．６％（４９／６０），４３．３％（２６／６０）及     

小凹蛋白与内皮型一氧化氮合成酶在大鼠肝硬化组织中的表达

目的探讨小凹蛋白Caveolin-1、内皮型一氧化氮合成酶eNOS在大鼠肝硬化组织中的异常表达及其意义。方法构建二甲基亚硝胺（DMN）致肝纤维化大鼠模型，在造模4周后观察肝纤维化程度。免疫组织化学染色检测30例大鼠肝硬化肝组织和30例正常大鼠肝组织中Caveolin-1和eNOS的细胞定位；Western Blot检测Caveolin-1和eNOS的蛋白表达水平变化。结果Caveolin-1和eNOS均主要分布于肝窦内皮细胞中，Caveolin-1在肝硬化组表达阳性率为90％，对照组为37％，两组比较差异有统计学意义（P〈0．05）；eNOS在肝硬化组表达阳性率为30％，对照组为66％，两组比较差异有统计学意义（P〈0．05）。Westem Blot检测Caveolin-1在肝硬化组织中较正常肝组织中表达明显增强；eNOS在肝硬化组织中呈低水平表达，较正常肝组织中表达明显减少。结论肝硬化肝窦内皮细胞中Caveolin-1的异常表达促进eNOS-Caveolin-1复合物的生成，结合形式的eNOS活性降低，导致NO合成减少，肝内血管阻力持续增加，从而导致了门静脉高压症的形成。     

慢性乙型肝炎和肝硬化中乙型肝炎病毒X抗原表达的分析

用基因工程技术由大肠杆菌合成重组乙型肝炎病毒（ＨＢＶ）Ｘ抗原（ＨＢｘＡｇ）并制备兔抗－ＨＢｘＩｇＧ以检测ＨＢｘＡｇ，合成一对以ＨＢＶＸ基因序列为模板的引物，用聚合酶链反应（ＰＣＲ）技术扩增ＨＢＶＤＮＡ。免疫组织化学和血清学方法分别用以分析肝病患者肝组织中的ＨＢｘＡｇ和血清标本中的ＨＢｘＡｇ、抗－ＨＢｘ。发现ＨＢｘＡｇ在慢性活动性肝炎（ＣＡＨ）患者肝组织中检出率为７２．７％，在肝炎后肝硬化（ＬＣ）中为９２．６％，而乙型肝炎核心抗原（ＨＢｃＡｇ）在ＬＣ中检出率为４７．８％。在ＣＡＨ、慢性迁延性肝炎和ＬＣ的血清中ＨＢｘＡｇ的阳性率分别为４４．４％、６６．６％和３３．３％，与ＨＢｅＡｇ的阳性率相似，而且在这些ＨＢｘＡｇ阳性的血清中可检出ＨＢＶＤＮＡ的存在，ＨＢｘＡｇ的表达与ＨＢＶ复制紧密相关，ＨＢｘＡｇ可能是一慢性ＨＢＶ感染的重要标志物。     

胃癌组织中基质金属蛋白酶及其组织抑制物的基因表达意义

目的观察胃癌组织及其癌变标本中基质金属蛋白酶（MMPs）及其金属蛋白酶组织抑制物（TIMPs）基因表达的变化。方法采用Northern—blot、原位分子杂交ISH的方法，对15例胃癌组织中MMP2、MMP9和TIMP1基因的mRNA表达水平进行了检测。结果胃癌癌组织MMP2、MMP9和TIMP1的基因表达水平明显高于正常组织。它们在胃癌癌组织和间质细胞中均有分布。结论组织中MMPs和TIMPs的异常表达有助于胃癌的早期诊断。     

Methodologic research on TIMP—1,TIMP—2 detection as a new diagnostic index for hepatic fibrosis and its significance

AIM: To set up a new method to detect tissue inhibitors of metalloproteinase-1 and -2(TIMP-1 and TIMP-2) in sera of patients with hepatic cirrhosis, and to investigate the expression and location of TIMP-1 and TIMP-2 in liver tissue of patients with hepatic cirrhosis, and the correlation between TIMPs in liver and those in sera so as to discuss whether TIMPs can be used as a diagnosis index of hepatic fibrosis. METHODS: The monoclonal antibodies (McAbs) of TIMP-1 and TIMP-2 were used to sensitize erythrocytes, and solid-phase absorption to sensitized erythrocytes (SPASE) was used to detect TIMP-1 and TIMP-2 in the sera of patients with hepatic cirrhosis. Meanwhile, with the method of in situ hybridization and immunohistochemistry, we studied the mRNA expression and antigen location of TIMP-1 and TIMP-2 in the livers of 40 hepatic cirrhosis patients with pathologic diagnosis. RESULTS: With SPASE, they were 16.4% higher in the acute hepatitis group, 33.3% higher in the chronic hepatitis group, and the positive rates were 73.6% and 61.2% respectively in sera of hepatic cirrhosis patients, which were remarkably higher than those in chronic hepatitis and acute hepatitis group (P<0.001). In 40 samples of hepatic cirrhosis tissues, all of them showed positive expression of TIMP-1 and TIMP-2 mRNA detected with immunohistochemistry or in situ hybridization (positive rate was 100%). Expression of TIMPs in different degrees could be found in liver tissue with cirrhosis. TIMPs were located in cytoplasm of liver cells of patients with hepatic cirrhosis. There was a significant correlation between serum TIMPs level and liver TIMPs level. CONCLUSION: SPASE is a useful method to detect the TIMP-1 and TIMP-2 in sera of patients with hepatic cirrhosis, and TIMP-1 and TIMP-2 can be considered as a useful diagnostic index of hepatic fibrosis, especially TIMP-1.     

Expression of leptin and leptin receptor during the development of liver fibrosis and cirrhosis.

Leptin is involved in the regulation of food intake and is mainly secreted by adipocytes. Major secretagogues are cytokines such as TNF-alpha or IL-1. Leptin in turn upregulates inflammatory immune responses. Elevated leptin serum levels have been detected in patients with liver cirrhosis, a disease frequently associated with elevated levels of circulating cytokines as well as hypermetabolism and altered body weight. Recently, leptin has been detected in activated hepatic stellate cells in vitro and an involvement of leptin in liver fibrogenisis has been suggested. The current study was designed to further clarify the role of leptin in liver disease by characterizing leptin and leptin receptor expression in the development and onset of experimental liver fibrosis. Liver fibrosis and cirrhosis was induced in rats by use of phenobarbitone and increasing doses of CCl (4). Leptin and leptin receptor mRNA expression was determined by semiquantitative RT-PCR, protein expression by Western blot analysis and localization of leptin and its receptor by immunohistochemistry. Normal liver tissue does not express leptin, but leptin receptor mRNA. Increasing levels of leptin mRNA were detected in fibrotic and cirrhotic livers correlated to the degree of fibrosis. Leptin receptor mRNA expression was not significantly altered in damaged livers. Increasing levels of leptin were detected in fibrotic and cirrhotic livers, whereas protein expression of the receptor remained unchanged. Throughout different stages of liver fibrosis, leptin immunoreactivity was localized in activated hepatic stellate cells only, whereas immunoreactivity for the receptor was mainly seen on hepatocytes. In conclusion, leptin is expressed at increasing levels in activated hepatic stellate cells in vivo, which may therefore be a source of increased leptin tissue and serum levels contributing to the pathophysiology and morphological changes of chronic liver disease.     

Evaluation of the diagnostic value of serum and tissue apoptotic cytokeratin-18 in patients with chronic hepatitis C

Background and study aimsHepatitis C virus (HCV) is considered the most common aetiology of chronic liver disease (CLD) in Egypt. The disease severity ranges from mild illness to cirrhosis and hepatocellular carcinoma. A role for apoptosis in liver damage caused by HCV chronic infection has been suggested. Cytokeratin 18 (CK-18) is the major intermediate filament protein in the liver and is a known caspase substrate in hepatocyte apoptosis. Therefore, we analysed the serum and tissue levels of CK-18 in patients with chronic HCV infection to evaluate its role in hepatocyte apoptosis. We also correlated CK-18 expression with the severity of hepatic pathology.Patients and methodsThis study examined 80 Egyptian patients with liver disease. There were 69 patients with chronic hepatitis C and 11 patients with hepatitis C-induced cirrhotic changes. Fifteen healthy controls were also included in the study. The levels of CK-18 fragment were quantified in paired serum and liver biopsy samples.ResultsThe serum and tissue CK-18 levels were reduced in chronic HCV patients compared to early cirrhosis patients. This result indicates that serum levels of CK-18 and the hepatic expression of CK-18 might play an important role in disease progression. The serum and tissue levels of CK-18 were significantly increased and directly correlated with inflammation severity, stage of fibrosis, and ALT levels in the chronic HCV group and the cirrhotic liver group. There was no significant difference in viral load between patient cohorts.ConclusionThe serum level and the hepatic expression of CK-18 are related to disease activity and are directly correlated with METAVIR scoring. This result suggests that serum CK-18 levels may be useful for monitoring disease activity in chronic HCV and liver cirrhosis patients.     

Expression of angiotensin II type 1 receptor in human cirrhotic livers: Its relation to fibrosis and portal hypertension.

Angiotensin II (ANG-II) and its receptor (AT1) have been potential targets of therapy for liver cirrhosis. However, AT1 expression in human cirrhotic livers has not been clarified. We studied AT1 and ANG-II generating enzymes in human autopsy (20 cirrhotics and 20 normal controls) and biopsy (10 cirrhotics) livers. AT1 immunoreactivity in tissue sections was quantified by computer-aided morphometry. AT1 protein and mRNA levels were assessed by Western blotting and real-time polymerase chain reaction. Concerning ANG-II generating system, angiotensin-converting enzyme (ACE) and mast cell chymase were examined. AT1 expression was seen not only in vascular smooth muscle cells, but also in activated stellate cells/myofibroblasts and liver parenchymal cells. AT1-positive vessels and myofibroblasts were significantly increased in fibrous septa of cirrhosis, although overall hepatic AT1 expression was reduced in the cirrhotic livers compared with the controls. Augmentation of AT1-positive vessels was related to severity of portal hypertension. Expressions of ACE and chymase were enhanced in the cirrhotic livers. These results suggest that hepatic AT1 expression is shifted to and concentrated in vessels and myofibroblasts in cirrhotic settings, and increased ANG-II generation by ACE and chymase contributes to portal hypertension and liver fibrosis via binding to AT1 expressed on vessels and myofibroblasts.     

Diagnostic potential of serum matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 as non-invasive markers of hepatic fibrosis in patients with HCV related chronic liver disease

As chronic liver disease progresses, an imbalance occurs between synthesis and breakdown of extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are involved in degrading ECM while tissue inhibitors of metalloproteinases (TIMPs) prevent their fibrolytic action. In the present study, serum levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were investigated as non-invasive parameters for the diagnosis of hepatic fibrosis in patients with HCV related chronic liver disease. Their diagnostic potential was evaluated in comparison to hepatic histology and standard liver function tests. A sandwich enzyme immunoassay technique was used to study circulating values of MMP-2 and TIMP-1 in forty-one patients with HCV antibodies in their sera (27 patients with biopsy ascertained chronic hepatitis C and 14 patients with histologically proven liver cirrhosis. Hepatic histology was evaluated using the hepatitis-activity-index according to Ishak et al. (1995), quantifying separately inflammatory activity and fibrosis. Ten healthy individuals were also included in the study as controls. Serum levels of MMP-2 were similar in controls and in chronic hepatitis C patients with (n = 15) and without (n = 12) fibrosis, but increased significantly in cirrhosis. TIMP-1 serum values showed a steady increase from normal controls to chronic hepatitis C without fibrosis, hepatitis C with fibrosis, and cirrhosis. The diagnostic potential of MMP-2 to detect fibrosis was low with a sensitivity of 7% and a diagnostic efficiency of 56%. The diagnostic potential of circulating MMP-2 to detect cirrhosis was higher with a sensitivity of 83% and a specificity of 96% resulting in a diagnostic efficiency of 92%. Serum TIMP-1 values detected fibrosis with a sensitivity of 67% and a specificity of 69% resulting in an efficiency rate of 70%. TIMP-1 values detected cirrhosis with 100% sensitivity but only 75% specificity. The diagnostic potential of circulating TIMP-1 was higher than that of serum ALT, AST or albumin values. In conclusion, serum values of MMP-2 and TIMP-1 are able to detect cirrhosis with a high sensitivity. Moreover, TIMP-1 values can detect fibrosis with comparable efficiency. Regular determinations of both TIMP-1 and MMP-2 in patients with chronic hepatitis C may be used as indicators of increasing fibrosis and the development of cirrhosis.     

Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation

BACKGROUND: The alpha isotype of actin expressed by hepatic stellate cells reflects their activation to myofibroblast-like cell and has been directly related to experimental liver fibrogenesis, and indirectly to human fibrosis in chronic liver disease. AIMS: To evaluate the changes in distribution and percentage of alpha-smooth muscle actin-positive hepatic stellate cells and the correlation with the degree of the fibrosis in cirrhotic livers, as well as in patients with recurrent HCV chronic hepatitis after liver transplantation. METHODS: Human liver biopsies were divided in four groups: (1) normal livers obtained from cadaveric liver donors (n=35), (2) cirrhosis post-HBV hepatitis (n=11), (3) cirrhosis post-HCV hepatitis (n=10), and (4) post-transplant recurrent HCV chronic hepatitis (n=13). Samples were stained with anti-alpha-smooth muscle actin antibody by immunoperoxidase method and semi-quantitatively evaluated. Liver fibrosis was assessed from specimens stained with Masson's trichrome and quantified by computer image analysis. RESULTS: The percentage of alpha-smooth muscle actin-positive hepatic stellate cells was significantly higher in the HBV cirrhosis, HCV cirrhosis and post-transplant HCV recurrent hepatitis groups (36.1+/-15.2, 23.8+/-19.7 and 27.8+/-16.4%, respectively) compared to the liver donor group (2.9+/-4.0%). The alpha-smooth muscle actin-positive hepatic stellate cells to fibrous tissue ratio were significantly higher in the post-transplant recurrent HCV hepatitis group (2.36+/-1.12) compared to both the donor livers and the HCV cirrhosis groups (0.74+/-1.09 and 1.03+/-0.91, respectively). The alpha-smooth muscle actin-positive hepatic stellate cell percentage and fibrosis correlated positively in the post-transplant recurrent HCV hepatitis group and negatively in the HCV cirrhosis group. No difference in the immunohistochemical and morphometrical variables was found between the HCV cirrhosis and HBV cirrhosis groups. CONCLUSIONS: These results indirectly confirm that, in vivo, alpha-smooth muscle actin expression is a reliable marker of hepatic stellate cells activation which precedes fibrous tissue deposition even in the setting of recurrent HCV chronic hepatitis after liver transplantation, and it could be useful to identify the earliest stages of hepatic fibrosis and monitoring the efficacy of the therapy. In the presence of advanced cirrhosis other factors, rather than alpha-smooth muscle actin-positive hepatic stellate cells, may sustain fibrosis deposition.     

Immunohistochemical study on tissue inhibitors of metalloproteinases in normal and pathological human livers

The localization of tissue inhibitor of metalloproteinases (TIMP) in normal and pathological livers was examined by immunohistochemistry using monoclonal antibodies at the light microscopic level. In normal liver, immunoreactive TIMP was detected in smooth muscle cells and endothelial cells of blood vessels, fibroblasts, bile duct cells and Kupffer cells, indicating that TIMP is likely to be a general element of the liver. Immunoreactivity was observed in newly-formed blood vessels, proliferating bile ductules, and fibroblasts in the expanded portal area and fibrous septa of chronic active hepatitis and cirrhosis. TIMP was strongly stained in the capsule of hepatocellular carcinoma. The intensity of the immunoreaction in the capsule was generally greater than that in cirrhotic liver apart from the tumor mass. In three of five cases with hepatocellular carcinoma, endothelial walls in contact with tumor cells were positive.