肝硬化患者外周血单个核细胞TLR4表达及其基因多态性的研究

目的：检测肝硬化及其不同Child分级患者外周血单个核细胞(PBMCs)Toll样受体4（TLR4）表达，分析TLR4基因Asp299Gly、Thr399Ile的多态性及其与肝硬化疾病发生的相关性。方法:分离110例汉族乙肝后肝硬化患者与健康对照者静脉抗凝血的PBMCs，流式细胞、实时荧光定量PCR及PCR-限制性片段长度多态性分别检测PBMCs TLR4蛋白、 mRNA及Asp299Gly、Thr399Ile基因多态性，分析TLR4基因多态性与肝硬化发生的相关性。结果:肝硬化组PBMCs TLR4阳性细胞率与mRNA表达正相关，均显著高于对照组(P<0.01)，且随着Child评分的升高而升高(P<0.01)；但220例受试对象均未检测到TLR4基因Asp299Gly、Thr399Ile的突变型。结论:乙肝后肝硬化患者PBMCs TLR4表达水平明显增高，与肝硬化损伤的严重程度相关；TLR4基因Asp299Gly、Thr399Ile多态性在中国汉族人群中出现频率低，且与肝硬化疾病的易感性可能无关。     

Dysregulation of blood lymphocyte subsets and natural killer cells in schistosomal liver cirrhosis and hepatocellular carcinoma

Immunological factors are important in the pathogenesis of a wide spectrum of hepatobiliary diseases. Using flow cytometry, we determined the changes in lymphocyte subsets and natural killer cells in 123 individuals (81 patients with liver disease and 42 healthy volunteers). The liver diseases included periportal fibrosis (PPF, 10 patients), liver cirrhosis (LC, 31 patients), and hepatocellular carcinoma (HCC, 40 patients). Schistosomiasis and viral hepatitis B and C were the putative etiological agents of liver diseases. Immunophenotyping by indirect immunofluorescence was conducted using monoclonal antibodies to CD3 (T-lymphocytes), CD4 (helper/inducer T-cells), CD8 (suppressor/cytotoxic T-cells), and CD57 (natural killer cells) cell surface markers. Immunophenotyping of PPF patients showed no significant changes in all markers compared with the healthy controls. However, there was a significant decrease ( P<0.01) in CD3 and CD4 T-cells, and a highly significant increase ( P<0.001) in CD57 T-cells in patients with LC or HCC. In addition, LC and HCC patients showed no significant change in CD8 T-cells compared with controls. In conclusion, the progression of liver diseases is associated with a dysregulation of cellular immune responses. T-lymphocytes and natural killer cells may play a role in the immunopathogenesis of liver cirrhosis and HCC.     

Morphometry of terminal hepatic veins

Summary In the present study, hepatic venous distribution per unit of liver surface area on normal wedge biopsies from man (n=11) and baboon (n=8) were analysed and compared. Terminal hepatic veins (THV - man:n=100; baboon:n=200) morphometric size variables were obtained with a Leitz ASM 68K morphometric equipment. THV, defined as hepatic veins up to 150 m in internal diameter (ID), in the centrolobular position and with sinusoidal openings, represented 84% and 74% of hepatic veins of man and baboon, respectively. Four or more THV were generally found on 8 mm² of liver surface. Transversely sectioned THV selected by the ratio IDminimum/IDmaximum >0.67, was found to be only 25% of the total THV. In baboon, THV merge with other terminal veins and the interlobular veins present sinusoidal inlets. The baboon THV wall surface (WS) and wall thickness (WT) values were higher than in man. Positive correlations between the number of mesenchymal cells (Mc) in the vein wall and wall surface of terminal hepatic veins (man: r= 0.79; baboon: r=0.83) and between wall surface and internal surface (IS) (man: r=0.80; baboon: r=0.72) were found. Two ratios were selected as the most reliable parameters: (1) for the THV wall rim, wall surface/internal surface (WS/IS - man: 0.43±0.16; baboon: 0.63±0.23), regarding transversely sectioned THV; and (2) for the evaluation of wall cell density (WS/Mc-man: 550±231; baboon: 558±183 m²/cell) as they did not depend on THV caliber.Dr. Porto was supported by a fellowship from MEC-CAPES, Brazil. A grant for morphometric equipment was obtained from the Fondation pour la Recherche Médicale and from the Societé d'Hépatologie Expérimentale, 77 rue Pasteur, Lyon, France     

Impaired renal function due to raised intraabdominal pressure

A 19-year-old male developed renal failure after a laparotomy for liver trauma (urinary output of 30 ml/h, plasma creatinine 220 mol/l). Surgical decompression of the abdomen was performed without any attempt at correcting the underlying pathology. This reduced the intraabdominal pressure (IAP) from 40 to 24 cm H₂O and resulted in a massive diuresis (530 ml/h). Twenty-four hours later the plasma creatinine peaked at 280 mol/l and then returned to within the normal range. This case report confirms that there is a direct relationship between IAP and renal function.     

Uptake and metabolism of 3H-adrenaline and 3H-noradrenaline by isolated hepatocytes and liver slices of the rat

Summary Isolated rat hepatocytes were incubated with 0.05 mol/l or 0.2 mol/l ³H-(–)-noradrenaline or 0.05 mol/l ³H-(–)-adrenaline for 15 min and the content of amines as well as the formation of metabolites was measured.The removal Of both amines from the incubation medium was quantitatively similar, and mainly due to metabolism (which represented 96% of the removal of ³H-adrenaline and 98% of the removal of ³H-noradrenaline). O-methylation predominated for ³H-adrenaline: O-methylated and deaminated metabolites (³H-OMDA) and ³H-metanephrine (³H-MN) were the most abundant metabolites, accounting for 63% and 34% of total metabolite formation, respectively. Deamination predominated for ³H-noradrenaline: ³H-OMDA and ³H-dihydroxymandelic acid (³H-DOMA) were the most abundant metabolites, representing respectively 56% and 36% of total metabolite formation. The following activities of monoamine oxidase and catechol-O-methyl transferase were determined for ³H-noradrenaline: k_COMT 0.70±0.15 min^–1 and k_MAO 2.27±0.14 min^–1 In experiments with ³H-noradrenaline, inhibition of monoamine oxidase reduced the formation of ³H-OMDA and deaminated metabolites [³H-dihydroxyphenylglycol (³H-DOPEG) and ³H-DOMA] and increased the formation of ³H-normetanephrine (³H-NMN). Inhibition of catechol-O-methyl transferase, On the Other hand, decreased ³H-NMN and increased ³H-DOPEG formation. When both enzymes were inhibited, the formation of all metabolites was strongly reduced but surprisingly there was no accumulation of ³H-amines in the cells, as the cell: medium ratio for ³H-noradrenaline or ³H-adrenaline was about unity. In experiments with either ³H-noradrenaline or ³H-adrenaline, specific inhibitors of either uptake, or uptake₂ produced discrete effects, slightly decreasing the formation of ³H-OMDA and ³H-NMN or ³H-MN, and having no effect on ³H-amine content of the cells. Additional experiments were carried Out with rat liver slices incubated for 15 min with ³H-noradrenaline 0.2 mol/l. The pattern of metabolism of ³H-noradrenaline (³H-OMDA and ³H-DOMA were the most abundant metabolites) as well as the degree of metabolism of the amine removed from the incubation medium (91% of the removal) were similar to those of the isolated cells. Likewise, there was no accumulation of intact ³H-noradrenaline in the tissue. Moreover, the results obtained with enzyme inhibitors as wells as with uptake inhibitors were similar to those obtained with hepatocytes.In conclusion, isolated hepatocytes remove and metabolize catecholamines very efficiently, being one of the most active systems studied in this respect. Uptake₁ and uptake₂ are responsible for part of the removal of catecholamines by hepatocytes; the system(s) involved in the remaining removal seem(s) to be active, but possess(es) characteristics that do not allow us to characterize it (them) either as uptake₁ or uptake₂.Abbreviations COMT catechol-O-methyl transferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MAO monoamine oxidase - MN metanephrine - NMN normetanephrine - OMDA O-methylated and deaminated metabolites (i.e., MOPEG = 4hydroxy-3-methoxyphenylglycol and VMA = 4-hydroxy-3-methoxymandelic acid) Supported by Programa STRIDE (STRDA/P/SAU/259/92)PhD student with a grant from JNICT (Programa Ciência) Correspondence to: F. Martel at the above address     

Hepatic arterial variations and liver-related diseases of 100 consecutive donors

We prospectively studied anatomical variations and diseases of the liver in 100 consecutive donor operations during a period of 1 year. The normal arterial anatomy with a single hepatic artery (HA) from the celiac trunk was seen in 76% of all cases. Seven of twelve different major variations of the HA may be considered as rare, one of which cannot be found in the earlier literature. During harvesting, 6% of the livers were discarded, 3% on the basis of infection and 1% because of a polycystic disease. Two cases were rejected as the liver was found to be severely hypoperfused or hypoxic in an otherwise stable donor. Severe steatosis was macroscopically and histologically diagnosed in 3% of the cases, and in three donors a benign tumour was found in the liver or in the gall bladder. Two primarily nonfunctioning livers in the present series of 94 recipient operations were retrieved from this group of severely steatotic livers. As the donor liver was totally normal in only 2 out of 3 of the cases, the present study underlines the importance of searching for extremely variable anomalies of the HA and for liver-related diseases during organ harvesting.     

Prediction of hepatic graft viability before reperfusion: an analysis of effluent from porcine allografts

Rapid and reliable assessment of hepatic graft viability is important for successful orthotopic liver transplantation (OLTx). OLTx was performed in 11 pairs of pigs via a venovenous bypass. Six of these grafts were transplanted immediately (group A), while the other five were preserved in University of Wisconsin (UW) solution for 24 h and then transplanted (group B). All grafts were flushed with 300 ml of chilled (4°C) Ringer's lactate solution before reperfusion of the graft, when 20 ml of effluent from the graft was collected and the concentrations of ammonia, lactic acid, GOT, and LDH were measured. Four of the six pigs in group A survived longer than 3 days, while the other two pigs died of causes other than graft dysfunction. All five pigs in group B died either of hemoperitoneum or hemodynamic instability due to liver failure. The histology of postperfusion biopsies in group A showed minimal pathological changes, while the grafts in group B revealed moderate to severe ischemic injuries. Ammonia and lactic acid in the effluent of group B were significantly higher than those of group A (1511±216 vs 417±333 g/dl and 114.1±12.2 vs 91.4±12.2 mg/dl, respectively; P<0.05 in both cases). Before reperfusion, the rate of total adenine nucleotides in all of the substances in the graft, which were measured using high performance liquid chromatography (HPLC), inversely correlated with the ammonia levels in the effluent. We conclude that an analysis of the effluent, (i.e. the levels of ammonia and lactic acid), flushed from a hepatic graft before reperfusion could serve as a predictor of hepatic graft viability.     

检测Ⅳ型胶原蛋白直接双抗夹心法的建立与评价

目的 :建立血清 型胶原蛋白 (COL - )浓度测定的直接双抗夹心法 ,为临床诊断肝纤维化提供一种新的指标。方法 :采用本科室自制的 COL - 单克隆酶标抗体、多克隆抗体建立的直接双抗夹心法 ,同时采用日本试剂盒测定 6 0份正常和肝脏病人血清 COL- 浓度做对照比较。结果 :两种方法无显著性差异 (P>0 .0 5 ,t=0 .16 6 ) ;两法相关分析 ,γ=0 .834,回归方程为 Y=0 .5 9X+ 70 .874。结论 :直接双抗夹心法测定血清 COL- 操作简便、特异、准确 ,可作为辅助诊断肝纤维化的可靠指标 ,并可在临床推广应用     

人原发性肝细胞癌核基质蛋白的研究

目的比较正常肝与原发性肝细胞癌（ＨＣＣ）核基质蛋白的异同,观察是否有ＨＣＣ特异性核基质蛋白的存在。方法用双向电泳方法比较了３例正常肝和８例ＨＣＣ核基质蛋白成分。结果正常肝和ＨＣＣ的核基质蛋白组成极为相似,但在ＨＣＣ中发现至少有４个ＨＣＣ的特异性核基质蛋白。其中以分子量为６２０００、等电点为５．３的蛋白最具代表性,它存在于所研究的８例ＨＣＣ中。其余３个ＨＣＣ特异性蛋白亦存在于大多数ＨＣＣ中。３例正常肝组织中未见有这４个ＨＣＣ特异性核基质蛋白。结论ＨＣＣ确实有ＨＣＣ特异性核基质蛋白的存在,它的发现可能会为ＨＣＣ的发生、发展、发病机理的研究提供一个新途径。