采用免疫型肝硬化大鼠建立慢加急性肝衰竭模型方法的研究

目的对人血清白蛋白免疫诱导型肝硬化大鼠给予不同急性打击，探索建立与人慢加急性肝衰竭组织病理表现相似、实用性、重复性好的实验模型、方法用人血清向蛋白免疫诱导建立肝硬化大鼠模型，至肝纤维化达4级时随机分组，组一（n=6）给予1．2g／kg的D．氨基半乳糖腹腔注射；组二（n=6）给予30mg／kg脂多糖尾静脉注射；组i（n=6）给予D-氨基半乳糖400mg／kg＋脂多糖100trg／kg同时腹腔注射；组四（n：4）继续给人血清白蛋白静脉注射，计算每组动物自然生存时间及死亡率、观察各脏器组织病理变化。结果组一5只大鼠给药后39～52h之内死亡，肝组织病理显示肝硬化基础上发生弥漫大小泡脂肪变性，仅见小片坏死灶；组二大鼠均存活良好，给药后3天后处死肝组织未见坏死性病变。组三5只大鼠在13～19h内死亡，肝脏病理表现为再生结节大块或亚大块坏死，肝细胞凋广明硅，增生的纤维间隔完整保留。结论对人血自蛋白免疫诱导型肝硬化大鼠给予D-氨基半乳糖／脂多糖联合急性攻击可建立慢加急性肝衰竭模型，而单独给大剂量D-氨基半乳糖或脂多糖不能使肝硬化大鼠发生肝脏大块或亚大块坏死。     

大鼠慢加急性肝衰竭模型肝脏组织病理学特征研究

目的 探讨大鼠慢性肝病基础上诱导的急性肝衰竭(慢加急性肝衰竭)模型肝脏组织病理学动态特征.方法 购入雄性SD大鼠,以50%四氯化碳植物油溶液腹腔注射,每3天1次,连续3个月(第一个月1.5 mL/kg体质量,第2、3个月 2.0 mL/kg体质量),建立大鼠肝硬化模型,对照组腹腔注射植物油溶液.肝硬化模型成立后,腹腔注射2 g/kg体质量D-氨基半乳糖(D-Galactosamine,D-Gal),建立肝硬化基础急性肝衰竭模型,并动态进行病理染色及电镜观察.结果 大鼠慢加急性肝衰竭模型制备良好,所有大鼠出现明显的腹水及黄疸症状,出现假小叶结构及细胞凋亡等.D-Gal急性攻击后,短时间内出现组织水肿、脂肪变性明显,大片嗜酸性变性.电镜上开始胞浆疏松、亚细胞器变形、糖原减少等,逐渐出现细胞器变少、异性结构、核固缩等现象.最终整个细胞器形态结构破坏,碎屑样小体出现及细胞溶解坏死.结论 肝硬化基础上药物急性攻击时除了细胞凋亡外,中毒所引起的溶解坏死后期参与大量肝细胞死亡.     

促肝细胞生长素颗粒剂对大鼠急性肝衰竭的防护作用(摘要)

目的:探讨促肝细胞生长素颗粒剂(促肝康)对大鼠急性肝衰竭的防护作用。方法:用D-氨基半乳糖(D-GalN)和内毒素脂多糖(LPS)制备大鼠急性肝衰竭模型,检测血清ALT、AST、TBil及肿瘤坏死因子α(TNFα)、内毒索(ET)的变化,并观察光镜下肝组织的病理改变。结果:模型组ALT、AST、     

内毒素性肝衰竭大鼠TLR4mRNA表达、血清肿瘤坏死因子-α及肝细胞凋亡的动态变化

目的 观察急性内毒素性肝衰竭大鼠肝组织中TLR4mRNA表达变化规律及其与血清肿瘤坏死因子-α水平、肝细胞凋亡的关系。方法雌性Wistar大鼠给予D氨基半乳糖／脂多糖同时腹腔注射，计算动物死亡率及生存时间，动态观察给药后4、8、12h肝功能、血清肿瘤坏死因子-α、肝组织TLR4mRNA表达及病理变化，以TUNEL法检测原位细胞凋亡，计算凋亡指数。结果80％大鼠死于急性肝衰竭，平均生存时间15．6h±1．8h，病理表现为肝脏大块或亚大块坏死。给药后4、8、12h血清TNF—α增高含量及肝细胞凋亡均增加，血清TNF—α变化早于肝细胞凋亡指数的增加，肝组织TLR4mRNA的表达与血清TNF—α含量呈正相关（r=0．709，P=0．000）。结论 内毒素通过单核吞噬系统TLR4介导TNF—α大量产生，激活炎症级联反应并诱导肝细胞凋亡是内毒素性肝衰竭的重要病理机制之一，阻断肝内外单核吞噬系统TLR4介导的的生理学作用，可能会对内毒素性肝衰竭起到一定防治作用。     

茵陈蒿汤上调树突状细胞Axl抑制慢加急性肝衰竭大鼠肝脏树突状细胞凋亡

目的:研究茵陈蒿汤对慢加急性肝衰竭(ACLF)模型大鼠肝内树突状细胞(DC)凋亡的影响及其与Axl信号的关系。方法:采用牛血清白蛋白致敏建立免疫性肝纤维化大鼠模型,然后用D-氨基半乳糖加脂多糖攻击建立ACLF模型,同时给予茵陈蒿汤灌胃治疗;观察攻击前及攻击后1h、12h其血清肝功能及肝脏组织病理学变化,应用CD11c/CD123+TUNEL凋亡双染色、CD11c/CD123+Axl-mRNA原位杂交双染色检测DC的凋亡及DCAxl的基因表达。结果:模型组大鼠表现为明显的肝纤维化基础上出现急性肝损伤,中药茵陈蒿汤组则炎症反应减轻,在攻击后1h、12h均显示有抑制树突状细胞凋亡的作用。正常组DC的Axl-mRNA表达水平较高,而模型组大鼠随着肝纤维化、肝衰竭的发展,Axl表达水平下降明显,茵陈蒿汤则具有上调DC的Axl表达的作用。DC凋亡指数与Axl-mRNA的表达呈线性负相关。结论:1肝内DC在由慢性肝病发展为ACLF进程中,其细胞凋亡增加可能是炎症活动不能控制,免疫修复障碍的重要原因。2Gas6/Axl通道是调控DC凋亡的重要机制。Axl信号可抑制DC凋亡。3茵陈蒿汤可上调Axl表达,抑制DC凋亡,这可能是其调节ACLF的免疫强度的重要机制。     

大鼠慢加急性肝衰竭模型建立的方法探讨

目的研究大鼠慢加急性肝衰竭模型的建立方法。方法以50％四氯化碳植物油溶液腹腔注射,每3天1次,连续3个月。其中,第1个月剂量为1.5mL／kg体重,第2、3个月剂量为2mL／kg体重。3个月后将大鼠随机分为3组,分别腹腔注射2g／kg体重D-氨基半乳糖、100μg/kg体重脂多糖联合0.5g／kg体重D-氨基半乳糖和5mL／kg体重50％四氯化碳植物油溶液。通过观察大鼠饮食和体重变化,测定大鼠血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和总胆红素（TBil）水平,以及观察大鼠肝组织病理形态改变来评价成模效果。结果大鼠腹腔注射四氯化碳植物油溶液3个月过程中,出现了不同程度的慢性肝损伤。其中,1个月后出现肝纤维化,2个月后表现为轻度肝硬化,3个月后出现明显肝硬化、腹水及黄疸症状。在前3个月的基础上再给予上述3种试剂后,短时间内诱导急性肝衰竭。结论四氯化碳腹腔注射3个月后大鼠出现明显肝硬化。在此基础上分别给予D-氨基半乳糖、脂多糖联合D-氨基半乳糖、四氯化碳,均可成功诱导肝硬化基础上的急性肝衰竭。     

D-氨基半乳糖/脂多糖诱导急性肝衰竭大鼠肝细胞凋亡的电镜观察

目的通过透射电镜观察急性肝衰竭大鼠肝脏超微结构,以了解肝细胞凋亡的形态学变化。方法给予雌性Wistar大鼠D-氨基半乳糖/脂多糖联合腹腔注射,计算其死亡率及生存时间,动态观察给药后4、8、12小时肝功能和组织病理学变化,并以TUNEL法检测和电镜观察原位细胞凋亡。结果实验组80%大鼠死于急性肝衰竭,平均生存时间为15.6±1.8小时。给药后4小时电镜观察,发现肝细胞线粒体肿胀、内质网扩张,胞浆包含体形成,部分细胞核呈早期凋亡表现,细胞内凋亡小体形成;8小时时,肝细胞凋亡明显增多并呈不同阶段表现,凋亡肝细胞内线粒体病变程度不一;TUNEL检测给药后8小时肝细胞,凋亡指数达高峰,与电镜观察结果一致。结论肝细胞凋亡为D-氨基半乳糖/脂多糖致急性肝衰竭大鼠肝细胞的主要病理形态学表现,这可能为该类型肝衰竭的主要病理学变化基础。     

糖皮质激素对慢加急性肝衰竭大鼠CD163及细胞因子的影响及意义

目的:研究糖皮质激素(地塞米松)对慢加急性肝衰竭大鼠模型CD163分子及促炎与抗炎因子的影响。方法:将30只无特殊病原体(SPF)级雄性Wistar大鼠分为3组,正常组与模型组各6只,治疗组18只。除正常组外,其余各组大鼠通过腹腔注射50%四氯化碳植物油溶液8周及D-氨基半乳糖(D-Gal)和脂多糖(LPS)联合冲击制备慢加急性肝功能衰竭模型。治疗组按地塞米松治疗时间不同分为不同3个时段:造模后0小时、造模后4小时、造模后8小时。各组于造模后24小时处死大鼠,用自动生化仪检测其血清ALT、AST、TBil,常规HE染色观察大鼠肝脏病理改变;实时定量PCR检测其肝组织中CD163分子水平;ELISA法检测其血清TNF-α及IL-10水平。结果:大鼠慢加急性肝衰竭模型复制成功,其肝组织纤维化基础上出现片状坏死,炎细胞浸润明显,肝脏生化指标显著升高,血清TNF-α及IL-10水平明显升高。造模后0小时地塞米松治疗后大鼠肝组织学明显好转,生化指标及血清TNF-α水平比模型组大鼠下降,而血清IL-10及肝组织中CD163分子表达水平升高,与模型组相比,差异有统计学意义(P<0.05);造模后4小时与8小时治疗组大鼠上述指标与模型组相比,差异无统计学意义。结论:慢加急性肝衰竭大鼠早期应用地塞米松可平衡促炎因子与抗炎因子,同时也促进了肝组织CD163分子的表达,对肝脏有保护作用。     

大黄对急性肝衰竭大鼠肝细胞凋亡的影响

目的 探讨大黄对急性肝衰竭大鼠肝细胞凋亡的影响及可能机制.方法 Wistar大鼠随机分为4组:正常对照组、模型组、大黄组和促肝细胞生长素(PHGF)组.大黄组和PHGF组于造模前3d分别每天灌服大黄水煎液和皮下注射PHGF 1 ml/100 mg,正常组和模型组每天灌服0.9％氯化钠注射液1 ml/100 mg.采用D-氨基半乳糖(D-GalN)/内毒素脂多糖(LPS)腹腔注射制备大鼠急性肝衰竭模型,造模8h后开腹取大鼠肝组织作病理检查,HE染色光镜下观察大鼠肝组织病理学变化,应用TUNEL法检测肝细胞凋亡情况,并应用免疫组化法分别检测肝组织中Fas、FasL和caspase-3的表达.结果 D-GαlN和LPS可引起严重的急性肝坏死并有广泛的肝细胞凋亡,伴Fas、FasL和caspaSe-3在肝细胞中强烈表达,大黄能减轻肝组织损伤,并可降低肝细胞凋亡及Fas、FasL和caspase-3表达(P均＜0.05).结论 大黄可抑制急性肝衰竭大鼠肝细胞凋亡,其机制可能与减轻肝细胞Fas、FasL和caspase-3表达有关.     

赤芍承气汤对急性肝衰竭大鼠肝细胞凋亡的影响

目的：观察赤芍承气汤对内毒素脂多糖（LPS）致急性肝衰竭大鼠肝细胞凋亡的影响。方法：以内毒素（LPS）＋D－氨基半乳糖（D－GalN)）联合制备大鼠急性肝衰竭模型,模型组用生理盐水、治疗组用复方中药赤芍 承气汤分别灌胃,4天后开腹取鼠肝组织作病理检查,以HE染色观察大鼠肝组织病理学变化;以免疫组化法（SP法）检测Fas,FasL蛋白在大鼠肝 的表达情况,并观察赤芍承气汤对肝细胞凋亡的影响。结果：LPS＋D－GalN可引起严重的急性肝坏死并有广阔物肝细胞凋亡,伴Fas,FasL蛋白在肝细胞中强烈表达;肝细胞Fas／FasL阳性率,模型组为83％：92％,中药组为53％：50％,经统计学分析,P＜0．01;其阳性表达率随肝细胞坏死程度加重而增加,结论：赤芍承气汤可减轻肝细胞Fas／FasL的表达,抵抗内毒素诱发的肝细胞凋亡。     

Doxorubicin coupled to lactosaminated albumin: Effects on rats with liver fibrosis and cirrhosis

BACKGROUND: The conjugate of doxorubicin with lactosaminated human albumin has the potential of increasing the doxorubicin efficacy in the treatment of hepatocellular carcinomas expressing the asialoglycoprotein receptor. However, coupled doxorubicin also accumulates in the liver, which might damage hepatocytes. AIMS: To verify whether coupled doxorubicin impairs liver function in rats with liver fibrosis and cirrhosis. METHODS: Coupled doxorubicin was administered using the same schedule which exerted an antineoplastic effect on rat hepatocellular carcinomas (4-weekly injections of doxorubicin at 1 microg/g). Liver fibrosis/cirrhosis was produced by carbon tetrachloride (CCl4) poisoning. Liver samples were studied histologically. Serum parameters of liver function and viability were determined. RESULTS: In normal rats, administration of coupled doxorubicin neither caused microscopic changes of hepatocytes nor modified serum liver parameters. In rats with fibrosis/cirrhosis, although a selective doxorubicin accumulation within the liver followed coupled doxorubicin administration, the drug did not have a detrimental effect on the histology of the liver and, among serum liver tests, only alanine aminotransferase and aspartate aminotransferase levels were moderately modified. CONCLUSIONS: Coupled doxorubicin can be administered to rats with liver fibrosis/cirrhosis without inducing a severe liver damage. If further studies will confirm the efficacy and safety of this compound, coupled doxorubicin therapy may open a new perspective in the treatment of hepatocellular carcinoma.     

肿瘤坏死因子-α诱导肝细胞凋亡在暴发性肝衰竭中的作用

目的 研究肿瘤坏死因子－α（ｔｕｍｏｒ ｎｅｃｒｏｓｉｓ ｆａｃｔｏｒ－α,ＴＮＦ－α）诱导肝细胞凋亡细胞凋亡在暴发性肝衰竭中的作用机制。方法 分别注射脂多糖（ｌｉｐｏｐ ｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）和ＴＮＦ－α于Ｄ－氨基半乳糖（Ｄ－ｇａｌａｃｔｏｓａｍｉｎｅ,ＧａｌＮ）致敏的ＢＡＬＢ／ｃ小鼠造成暴发性肝衰竭模型,用脱氧核糖核酸转移酶介导的缺口原位末端标记（ｉｎ ｓｉｔｅ ｅｎｄ ｌａｂｅ     

Hepatocyte growth factor prevents endotoxin-induced lethal hepatic failure in mice.

Sepsis and endotoxemia are involved in the development of fulminant hepatic failure, the prognosis of which is extremely poor and the mortality is high, with no available effective therapy. Here, we report that hepatocyte growth factor (HGF) exerts potent antiapoptotic effects in vivo and effectively prevents endotoxin-induced fulminant hepatic failure in mice. The animals were intraperitoneally injected three times with 120 micrograms human recombinant HGF or saline 6 hours and 30 minutes before and 3 hours after an intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (GalN). Administration of LPS + GalN, without HGF, rapidly led to massive hepatocyte apoptosis and severe liver injury, and all mice died of hepatic failure within 8 hours. In contrast, administration of human recombinant HGF strongly suppressed extensive progress of hepatocyte apoptosis and the liver injury induced by LPS + GalN, and 75% of the HGF-treated mice survived. Moreover, HGF strongly induced Bcl-xL expression and blocked apoptotic signal transduction upstream of CPP32 (caspase-3) in the liver, thereby leading to inhibition of massive hepatocyte apoptosis. We suggest that HGF may well have the potential to prevent fulminant hepatic failure, at least through its potent antiapoptotic action.     

Hybrid bioartificial liver support in cynomolgus monkeys with D-galactosamine-induced acute liver failure

AIM: To evaluate a hybrid bioartificial liver support system (HBALSS) in cynomolgus monkeys with acute liver failure.METHODS: To establish a model of acute liver failure, 0.3 g/kg of D-galactosamine was injected intravenously into cynomolgus monkeys. Chinese human liver cells were introduced into a perfusion bioreactor to carry out hybrid bioartificial liver support treatment. Forty-eight hours after the injection, one group of cynomolgus monkeys received HBALSS care, and a second experimental group received no treatment. Clinical manifestations of all animals, survival time, liver and kidney functions and serum biochemistry changes were recorded. Simultaneous detection of the number, viability and function of hepatocytes in the hybrid bioartificial liver were also performed.RESULTS: Forty-eight hours after the injection of D-galactosamine, serum biochemistry levels were significantly increased, whereas albumin levels and the Fischer index were significantly reduced compared to baseline (all Ps < 0.05). Of the ten monkeys in the HBALSS treatment group, five survived, with an average duration of survival of 128 ± 3 h. All cynomolgus monkeys in the control group died, with a duration of survival of 112 ± 2 h. Survival time was significantly longer with HBALSS treatment (P < 0.05). Moreover, the number, viability and function of hepatocytes were maintained at a high level with HBALSS.CONCLUSION: The novel hybrid bioartificial liver plays a significant role in liver support by significantly reducing serum biochemistry levels and extending animal survival time.     

Toll样受体在肝衰竭发病中的作用研究进展

肝衰竭是多种病因引起的肝功能失代偿，其发病机制涉及肝细胞的坏死、凋亡、再生、肝脏纤维化等。Toll样受体（TLR）作为一种模式识别受体，参与了固有免疫反应和获得性免疫反应，在肝衰竭的发病中起重要作用。动物实验和临床研究均已证实TLR与肝细胞的坏死有关。脂多糖（LPS）、D-氨基半乳糖（D-GalN）、刀豆素A（ConA）、CpG寡脱氧核苷酸（CpG ODN）、多聚肌苷酸-多聚胞苷酸（Poly I：C）和对乙酰氨基酚（APAP）所致的肝损伤需要不同的TLR通路参与。TLR3、TLR4、TLR9参与了肝细胞的凋亡。TLR7对肝纤维化有保护作用。TLR基因多态性研究可以从宿主方面揭示肝衰竭的发病机制，为个体化诊疗提供依据。     

Effect of PGE1 on TNF-alpha status and hepatic D-galactosamine-induced apoptosis in rats

Prostaglandin E1 has hepatoprotective properties in several clinical and experimental models of liver dysfunction. Hepatotoxicity induced by D-galactosamine (D-GalN) is a suitable animal model of human acute hepatic failure. The aim of the study was to investigate if prostaglandin E1 (PGE1) protection against hepatic D-GalN-induced apoptosis was related to tumour necrosis factor-alpha (TNF-alpha) content in serum. This cytokine is associated with in vitro apoptosis and general inflammatory disorders. In this study, PGE1 was administered 30 min before D-GalN to rats. In other experiments, several doses of TNF-alpha were administered 15min after PGE1 to D-Ga1N-treated rats. Several parameters related to apoptosis and necrosis were measured by flow cytometry, gel electrophoresis, biochemical analysis, and optical and electron microscopy. Tumour necrosis factor-alpha was quantified by competitive enzyme-linked immunosorbent assay (ELISA). PGE1 by itself did not modify the cell cycle of hepatocytes and liver toxicity, but increased TNF-alpha in serum in comparison with the control group. D-Galactosamine increased the percentage of hepatocytes in apoptosis and in the S phase of the cell cycle, and decreased those in G0/G1. Such an increase of hepatocytes in apoptosis was correlated with a higher number of apoptotic bodies and DNA fragmentation in liver than control samples. Also, D-GalN increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase and TNF-alpha in serum compared with the control group. Pre-administration of PGE1 to D-GalN-treated rats reduced all the parameters of apoptosis and necrosis in liver, and increased additionallyTNF-alpha content in serum. In those experiments where low doses of TNF-alpha were administered to PGE1 and D-GalN-treated rats an inverse relationship appeared between TNF-alpha and ALT content in serum. In conclusion, the protective effects of PGE1 on D-GalN-induced apoptosis may be linked to its capacity to modulate cell division and/or its immunomodulatory activity. In this sense, our experimental results suggest that TNF-alpha could be involved in protection or exacerbation of liver damage in relation to the pathophysiological status of the liver.     

Expression and distribution of prolactin receptor in normal, fibrotic, and cirrhotic human liver.

Liver cirrhosis is often associated with elevated levels of prolactin (PRL). This is commonly attributed to impaired hepatic metabolism of estrogens. However, there is evidence suggesting that PRL may be an important factor in hepatic tissue regeneration. To investigate the role of PRL in the pathogenesis of liver cirrhosis, we used RT-PCR and immunhistochemical staining to analyze changes in the expression and the histological distribution of the prolactin receptor (PRLR) in normal, fibrotic and cirrhotic hepatic tissue. Liver tissue was obtained from 29 surgically explanted human livers. The histological examination demonstrated normal liver tissue (n=9) as well as different grades of fibrosis (n=10) and cirrhosis (n=10). In liver cirrhosis and fibrosis, PRLR-mRNA was expressed at a higher level compared to normal liver specimens. Immunohistochemical staining of normal liver tissue demonstrated homogeneous distribution of the PRLR in the hepatocytes and in the epithelial cells of the bile ducts. This pattern of distribution was lost in fibrosis, where an accumulation of the PRLR was observed in the damaged hepatocytes. As no PRL-mRNA was detectable in normal, fibrotic or cirrhotic tissue, PRL does not act through autocrine or paracrine mechanisms. These data confirm previous results, which we obtained using an animal model for experimental liver cirrhosis in rats suggesting a metabolic function of PRL in normal liver and a regenerative function in fibrotic and cirrhotic liver. In conclusion, PRL might be involved in the pathogenesis of liver cirrhosis.