大鼠肝纤维化过程中肝内成纤维细胞的免疫组化研究

目的肝脏内存在有两种成纤维细胞,一种是肝星形细胞(hepatic stellate cell,HSC),一种是门脉区域的成纤维细胞。用免疫组化方法对这两种细胞群在肝纤维化(hepatic fibrosis,HF)中活化后的细胞形态进行组织学鉴别。方法Wistar系雄性鼠用于实验动物模型制作:CCL4腹腔注射和胆总管结扎(bile duct ligation,BDL)诱导肝纤维化动物模型,用SABC法来检测。一次抗体分别为GFAP、α-SM、Desmin、Vinculin。结果CCl4腹腔注射后48h中央静脉周围的星形细胞在GFAP、α-SMA、Desmin、Vinculin染色呈强阳性;另外,BDL96h后门脉区域成纤维细胞在α-SMA、Vinculin染色中显示阳性,而在GFAP、Desmin染色中未发现有变化。结论α-SMA、Vinculin在肝星形细胞和门脉区域成纤维细胞有相同的阳性结果,GFAP、Desmin被证明能够用于区别有活性的肝星形细胞和门脉区域的成纤维细胞。     

Hepatic structural correlates of liver fibrosis: a morphometric analysis

Liver fibrosis is an important feature of many liver diseases, and the assessment of fibrosis is essential for diagnosis and prognosis. Liver needle biopsy, however, tends to sample preferentially the soft parenchyma, which poorly reflects the true extent of fibrosis. Therefore, we have searched for capsule and parenchymal features that are correlated with the fibrosis. To test for these correlations, we used a morphometric analysis of a rat model of liver fibrosis. We demonstrated that, of the variables examined, capsule thickness was the best correlate of liver fibrosis. Our results also showed that there were parenchymal structures that underwent changes that were well-correlated with the development of fibrosis in the liver. These changes were identified as increases in hepatocyte nucleus, cytoplasm, and mitochondria. These facts suggest that, at least in a rat model of fibrosis, capsule thickness is a useful parameter by which to assess liver fibrosis and that specific and apparently adaptive parenchymal changes are well-correlated to fibrosis.     

Cystic fibrosis liver sialyltransferase

The activity of sialytransferase with regard to the glycoprotein substrates asialofetuin and asialo-ovine submaxillary mucin was determined in normal, pathological control, and cystic fibrosis liver homogenates. Cystic fibrosis and pathological livers have about 40% of the average normal specific activity for sialytransferase. Several properties of cystic fibrosis sialytransferase were investigated and compared to those of the normal liver enzyme (Alhadeff et al. 1977). The pH optima curves were similar, but cystic fibrosis sialyltransferase appears to be more thermolabile than the normal liver enzyme. Isoelectric focusing studies revealed that the three most basic forms of sialyltransferase which are found in normal livers are deficient or absent in most cystic fibrosis liver. The data suggest that altered glycoprotein-sialyltransferases may be present in cystic fibrosis livers, probably a secondary effect due to general liver pathology.     

Fractal and Fourier analysis of the hepatic sinusoidal network in normal and cirrhotic rat liver

The organization of the hepatic microvascular network has been widely studied in recent years, especially with regard to cirrhosis. This research has enabled us to recognize the distinctive vascular patterns in the cirrhotic liver, compared with the normal liver, which may explain the cause of liver dysfunction and failure. The aim of this study was to compare normal and cirrhotic rat livers by means of a quantitative mathematical approach based on fractal and Fourier analyses performed on photomicrographs and therefore on discriminant analysis. Vascular corrosion casts of livers belonging to the following three experimental groups were studied by scanning electron microscopy: normal rats, CCl(4)-induced cirrhotic rats and cirrhotic rats after ligation of the bile duct. Photomicrographs were taken at a standard magnification; these images were used for the mathematical analysis. Our experimental design found that use of these different analyses reaches an efficiency of over 94%. Our analyses demonstrated a higher complexity of the normal hepatic sinusoidal network in comparison with the cirrhotic network. In particular, the morphological changes were more marked in the animals with bile duct-ligation cirrhosis compared with animals with CCl(4)-induced cirrhosis. The present findings based on fractal and Fourier analysis could increase our understanding of the pathophysiological alterations of the liver, and may have a diagnostic value in future clinical research.     

Mononuclear cells in liver fibrosis

Fibrosis is a multicellular wound healing process, where myofibroblasts that express extracellular matrix components extensively cross-talk with other cells resident in the liver or recruited from the bloodstream. Macrophages and infiltrating monocytes participate in the development of fibrosis via several mechanisms, including secretion of cytokines and generation of oxidative stress-related products. However, macrophages are also pivotal in the process of fibrosis resolution, where they contribute to matrix degradation. T lymphocytes modulate the fibrogenic process by direct interaction with myofibroblasts and secreting cytokines. In general, Th2 polarized responses promote fibrosis, while Th1 cytokines may be antifibrogenic. NK cells limit the development of fibrosis and favor its resolution, at least in part via killing of fibrogenic cells. The possible role of NKT cells and B cells is emerging in recent studies. Thus, mononuclear cells represent a critical regulatory system during fibrogenesis and may become an appealing target for therapy.     

Cholesterol-induced stimulation of postinflammatory liver fibrosis

We studied the effect of high-cholesterol diet and factors inhibiting 3-hydroxy-3-methyl-glutaryl coenzyme A reductase on the development of liver fibrosis in C57Bl/6 mice with CCl₄-or zymosan-induced hepatitis. Feeding a high-cholesterol diet led to a sharp increase in collagen content in the liver tissue of animals with CCl₄-induced or zymosan-induced hepatitis. Atorvastatin and calcitriol produced less pronounced fibrogenic effects. Mevalonate partially prevented the development of cholesterol-induced fibrogenesis. High-cholesterol diet led to accumulation of oxysterols, cholesterol esters, and triglycerides and increased the expression of transforming growth factor-β₁ mRNA in liver tissue. Cholesterol-induced potentiation of the fibrogenic response is probably associated with transforming growth factor-β₁ induction due to accumulation of lipids and oxysterols in the liver. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 145, No. 6, pp. 638–641, June, 2008     

吡非尼酮抗肝纤维化机制

吡非尼酮是正在研发的新型广谱抗纤维化药物,目前抗肝纤维化治疗已进入Ⅱ期临床试验阶段,其作用机制与抑制脂质过氧化、减轻炎症反应、抑制肝星状细胞活化和增殖、调节细胞外基质的合成与降解有关.本文对吡非尼酮抗肝纤维化的研究现状及作用机制作一综述.     

Fractal geometric analysis of colorectal polyps

Colorectal polyps have a subjectively self-similar structure which suggests that these structures may have fractal elements and that the fractal dimension may be a useful morphometric discriminant. The fractal dimensions of images from haematoxylin and eosin-stained sections of 359 colorectal polyps (214 tubulovillous adenomas, 41 ‘pure’ tubular adenomas, 29 ‘pure’ villous adenomas, 68 metaplastic polyps, and 7 inflammatory polyps) were measured using a box-counting method implemented on a microcomputer-based image analysis system. Results were assessed using polychotomous logistic regression, confusion matrices, and kappa statistics. All examined polyps were shown to have a fractal structure in the range of scales examined. The fractal dimension was significantly different between different diagnostic categories (P<0·0001) and was a useful discriminant between these categories (kappa statistic 0·60 for the confusion matrix with size as the other variable). The fractal dimension did not shown any significant correlation with the grade of epithelial dysplasia (P>0·05). This study shows that colorectal polyps have a fractal structure over a defined range of magnification and Euclidean morphometric measurements will be invalid outside precisely defined conditions of resolution and magnification. The fractal dimension is a better way of quantitating the polyp shape and is a useful morphometric discriminant between diagnostic categories.     

慢性肝病肝剪切波速与血清肝纤维化标志物在肝纤维化诊断中的对比研究

目的 探讨慢性肝病肝剪切波速及血清肝纤维化标志物在肝纤维化分级诊断中的临床应用价值.方法 选择235例慢性肝病患者(肝纤维化组),其中男性179例,女性56例;年龄17～64岁,平均年龄36.3岁.40例健康对照者,其中男性25例,女性15例;年龄25～62岁,平均年龄43.2岁.先进行肝剪切波速和抽血实验室检查,然后在超声引导下穿刺活组织检查,以病理结果为标准,分析剪切波速及血清肝纤维化标志物在评价肝纤维化分级之间的关系.并将检查结果与健康对照者进行比较.结果 235例慢性肝病患者有215例进行肝穿刺活组织检查,除F0级肝病患者与健康对照组F0级之间剪切波速比较差异无统计学意义(P＞0.05)外,其余肝病各分组剪切波速随肝纤维化程度的增高而波速增快;且血清肝纤维化标志物含量亦随肝纤维化病理分期的增高而增高.结论 肝剪切波速及血清肝纤维化标志物对肝纤维化患者的病理分期有良好相关性,对肝纤维化的诊断及疗效观察有重要的临床应用价值.     

Severe liver fibrosis in argininosuccinic aciduria

Hepatomegaly is an important clinical finding in patients with argininosuccinic aciduria (a hereditary defect of the urea cycle enzyme, argininosuccinate lyase [argininosuccinase]). A severe degree of liver fibrosis, almost corresponding to cirrhosis, was observed in liver biopsy material obtained from a boy with this disorder. This observation is of interest in light of the fact that liver fibrosis or cirrhosis are hallmarks of many inheritable phenotypes, and especially of inborn errors of metabolism. Variable degrees of liver fibrosis are noted in other inborn defects of the urea cycle, eg, in ornithine transcarbamylase and carbamoylphosphate synthetase deficiencies. These findings appear to indicate that inheritable defects of urea synthesis may form a group of metabolic disorders prone to cause hepatic fibrosis, or even cirrhosis, as shown in our patient.     

Respective roles of porto-septal fibrosis and centrilobular fibrosis in alcoholic liver disease

In alcoholic liver disease, fibrosis classically begins around the centrilobular veins, while portal tract fibrosis is described as inconstant and septal fibrosis is a late event. The aim of this study was to compare the roles of centrilobular fibrosis (CLF) and portal tract/septal fibrosis (PTF) especially in alcoholic chronic liver disease. One hundred and sixty patients with alcoholic chronic liver disease and 83 controls with viral chronic hepatitis were included. The PTF score, derived from the Metavir score, CLF and the area of fibrosis, assessed by image analysis, were evaluated on liver biopsies in addition to blood markers of fibrosis. The correlation between the PTF score and the area of fibrosis was higher in alcoholic liver disease (r = 0.87, p < 10(-4)) than in viral chronic hepatitis (r = 0.66, p < 10(-4)). The PTF score correlated with the CLF score (r = 0.92, p < 10(-4)), serum hyaluronate (r = 0.76, p < 10(-4)), and the prothrombin index (r = -0.77, p < 10(-4)). Multiple regression analyses showed that the area of fibrosis was explained only by the PTF score and not by the CLF score. PTF appears more frequent than CLF in alcoholic chronic liver disease, suggesting that PTF may precede CLF. PTF is more responsible for the amount of fibrosis than CLF. The results of this study also validate the use of the Metavir fibrosis score in alcoholic chronic liver disease.     

Pathomorphology of congenital liver fibrosis in children

Pathomorphology of congenital liver fibrosis (CLF) was studied basing on histological evaluation of 42 primary and secondary liver biopsies obtained from 26 CLF children aged 1-13. CLF age-specific complications, their variants and incidence rate as well as morphogenesis are outlined, e. g. cholangiolitis and secondary cirrhosis which are not rare in 7-13-year-old children. Presented is CLF staging.     

Cenicriviroc治疗肝纤维化的实验研究

探讨Cenicriviroc(CVC)对硫代乙酰胺(TAA)所致大鼠肝纤维化的治疗作用。SD雄性大鼠66只被随机分为模型对照组和CVC治疗组。各组大鼠于同日开始腹腔注射TAA 30mg/Kg,3次/周,连续8周;另设空白对照组。治疗组分别在TAA诱导肝纤维化不同时间,按30mg·kg-1·d-1灌胃给药,包括:与TAA同日开始给CVC,连续8周(1B组);TAA腹腔注射4周后,给予CVC,连续4周(2B组)和TAA腹腔注射8周停用后,再给予CVC,4周(3B组)。治疗结束后,检测血清中生化指标,留取肝组织,行HE和苦味酸天狼星红溶液染色,观察肝脏病理学改变,并进行肝纤维化定量分析。同时,以qRT-PCR检测CVC对培养肝星状细胞(HSC)表达α-平滑肌肌动蛋白(α-SMA)和胶原蛋白I(collagen I)的直接作用。与模型组比,早、中期CVC治疗(1B组和2B组)显著降低肝纤维化大鼠增高的肝脏系数(P0.05)和血清中异常升高的天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)(P0.01),病理组织学检查结果表明,1B组和2B组大鼠肝纤维组织增生和肝组织炎症活动度较模型组均有显著的改善(P0.05),其肝组织中胶原纤维面积显著降低(P0.01),但是CVC对已形成的肝硬化(3B组)无显著改善作用。体外实验显示,CVC可以直接抑制HSC胶原蛋白I mRNA表达(P0.05),但α-SMA mRNA表达与对照组无明显差异。CVC具有明显抑制肝纤维化形成作用,但对已形成的肝硬化缺乏逆转作用。     

Hepatic stellate cells and liver fibrosis

Substantial evidence now exists to recognize hepatic stellate cells (HSCs) as the main matrix-producing cells in the process of liver fibrosis. Liver injury of any etiology will ultimately lead to activation of HSCs, which undergo transdifferentiation to fibrogenic myofibroblast-like cells. Quantitative analysis of HSC activation by immunohistochemistry has been shown to be useful in predicting the rate of progression of liver fibrosis in some clinical situations. In the activation process, transforming growth factor beta is thought to be the main mediator of fibrogenesis and platelet-derived growth factor is the major inducer of HSC proliferation. Different platelet-derived growth factor and transforming growth factor beta inhibitors have been shown to effectively prevent liver fibrosis in animal models and represent promising therapeutic agents for humans.     

The role of adipokines in liver fibrosis

Liver fibrosis is a dynamic process consisting of the chronic activation of the wound healing reaction in response to reiterated liver damage, leading to the excessive deposition of fibrillar extracellular matrix into the liver and eventually, if the cause of injury is not removed, to liver cirrhosis. The term "adipokines" identifies a group of polypeptide molecules secreted primarily by adipose tissue, which exert local, peripheral and/or central actions. Additionally to their well-established role in controlling adipose tissue physiology, adipokines have been shown to be involved in different obesity-related diseases, such as hypertension, atherosclerosis and type 2 diabetes. Accumulating data demonstrate that obesity and insulin resistance are associated with a more severe and faster progression of the fibrogenic process in different chronic liver diseases. Therefore, numerous recent studies have analyzed the role played by adipokines in the hepatic wound healing process, identifying novel roles as modulators of liver pathophysiology. This review summarizes the more significant and recent findings concerning the role played by adipocyte-derived molecules, such as leptin, adiponectin and resistin, in the liver fibrogenic process. The actions of different adipokines on the biology of liver resident cells, as well as their effects in different animal models of liver injury are discussed. The variations in the circulating levels and in the intrahepatic expression of these molecules occurring in patients with different chronic liver diseases will be also analyzed.     

Toward surface quantification of liver fibrosis progression

Monitoring liver fibrosis progression by liver biopsy is important for certain treatment decisions, but repeated biopsy is invasive. We envision redefinition or elimination of liver biopsy with surface scanning of the liver with minimally invasive optical methods. This would be possible only if the information contained on or near liver surfaces accurately reflects the liver fibrosis progression in the liver interior. In our study, we acquired the second-harmonic generation and two-photon excitation fluorescence microscopy images of liver tissues from bile duct-ligated rat model of liver fibrosis. We extracted morphology-based features, such as total collagen, collagen in bile duct areas, bile duct proliferation, and areas occupied by remnant hepatocytes, and defined the capsule and subcapsular regions on the liver surface based on image analysis of features. We discovered a strong correlation between the liver fibrosis progression on the anterior surface and interior in both liver lobes, where biopsy is typically obtained. The posterior surface exhibits less correlation with the rest of the liver. Therefore, scanning the anterior liver surface would obtain similar information to that obtained from biopsy for monitoring liver fibrosis progression.     

Quantitative analysis of liver fibrosis in rats with shearwave dispersion ultrasound vibrometry: Comparison with dynamic mechanical analysis

Ultrasonic elastography, a non-invasive technique for assessing the elasticity properties of tissues, has shown promising results for disease diagnosis. However, biological soft tissues are viscoelastic in nature. Shearwave dispersion ultrasound vibrometry (SDUV) can simultaneously measure the elasticity and viscosity of tissue using shear wave propagation speeds at different frequencies. In this paper, the viscoelasticity of rat livers was measured quantitatively by SDUV for normal (stage F0) and fibrotic livers (stage F2). Meanwhile, an independent validation study was presented in which SDUV results were compared with those derived from dynamic mechanical analysis (DMA), which is the only mechanical test that simultaneously assesses the viscoelastic properties of tissue. Shear wave speeds were measured at frequencies of 100, 200, 300 and 400 Hz with SDUV and the storage moduli and loss moduli were measured at the frequency range of 1–40 Hz with DMA. The Voigt viscoelastic model was used in the two methods. The mean elasticity and viscosity obtained by SDUV ranged from 0.84 ± 0.13 kPa (F0) to 1.85 ± 0.30 kPa (F2) and from 1.12 ± 0.11 Pa s (F0) to 1.70 ± 0.31 Pa s (F2), respectively. The mean elasticity and viscosity derived from DMA ranged from 0.62 ± 0.09 kPa (F0) to 1.70 ± 0.84 kPa (F2) and from 3.38 ± 0.32 Pa s (F0) to 4.63 ± 1.30 Pa s (F2), respectively. Both SDUV and DMA demonstrated that the elasticity of rat livers increased from stage F0 to F2, a finding which was consistent with previous literature. However, the elasticity measurements obtained by SDUV had smaller differences than those obtained by DMA, whereas the viscosities obtained by the two methods were obviously different. We suggest that the difference could be related to factors such as tissue microstructure, the frequency range, sample size and the rheological model employed. For future work we propose some improvements in the comparative tests between SDUV and DMA, such as enlarging the harmonic frequency range of the shear wave to highlight the role of viscosity, finding an appropriate rheological model to improve the accuracy of tissue viscoelasticity estimations.     

The pediatric NAFLD fibrosis index: a predictor of liver fibrosis in children with non-alcoholic fatty liver disease

Background  

Liver fibrosis is a stage of non-alcoholic fatty liver disease (NAFLD) which is responsible for liver-related morbidity and mortality in adults. Accordingly, the search for non-invasive markers of liver fibrosis has been the subject of intensive efforts in adults with NAFLD. Here, we developed a simple algorithm for the prediction of liver fibrosis in children with NAFLD followed at a tertiary care center.