肝纤维化大鼠肝脏中组蛋白修饰水平的变化

目的:观察肝纤维化大鼠肝脏中组蛋白修饰的变化,并探讨其在肝纤维化发生发展过程中可能的作用。方法:雄性Wistar大鼠20只,随机分为正常对照组和肝纤维化组,其中肝纤维化组采用CCl_4皮下注射以制备大鼠肝纤维化模型,正常组注射等量植物油溶液。实验第8周末,股动脉放血处死大鼠,取2组血清,采用生化和放射免疫法测定血清肝功能指标丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST),以及肝纤维化标志物血清透明质酸(HA)、层粘连蛋白(LN)、Ⅳ型胶原(Col Ⅳ)和Ⅲ型前胶原(PCⅢ)的水平;取2组大鼠肝脏,测定肝脏指数;取肝组织常规固定,HE染色和Masson染色观察组织病理改变及胶原纤维沉积情况;Western blot检测2组大鼠肝脏组织中α-平滑肌肌动蛋白(α-SMA)和I型胶原(ColⅠ)表达情况,以及acH4K12、acH3K9、H3K4me2和H3K9me2修饰水平的变化。结果:与对照组相比,模型组大鼠肝脏指数及ALT、AST、HA、LN、ColⅣ和PCⅢ水平明显增高(P0.05);Western blot检测发现,与对照组比较,肝纤维化组大鼠肝组织的acH4K12修饰水平减少(P0.05),acH3K9和H3K9me2修饰水平及α-SMA和ColⅠ表达明显增加(P0.05),H3K4me2修饰水平的差异无统计学显著性。结论:肝纤维化大鼠肝脏中acH4K12、acH3K9和H3K9me2修饰水平改变可能与某些细胞外基质代谢相关基因转录调控有关,从而参与了大鼠肝纤维化发生。     

大鼠腹腔注射人血清白蛋白免疫性肝纤维化模型的实验研究

目的 通过腹腔注射人血清白蛋白建立免疫损伤性大鼠肝纤维化模型。方法 雄性Wistar大鼠，体重110—120g，皮下多点注射人血清白蛋白（共4次。分别间隔14、10、10d）。10d后，腹腔注射人血清白蛋白（每周2次，共8周，剂量从5mg逐渐增至20mg）。在攻击前、攻击后15、30、60d和停止攻击后30、60、90、120d，分别留取肝组织和血清标本，进行肝病理检查和肝羟脯氨酸生化测定，采用放射免疫法测定肝纤维化血清指标透明质酸（HA）和层粘连蛋白（LN）含量。结果 攻击后。肝纤维化分级、肝羟脯氨酸含量、血清HA、LN均升高（P〈0．05）。随造模时间延长，肝纤维化分级逐渐加重（P〈0．05）、肝羟脯氨酸含量、血清HA逐渐升高（P〈0．01）。停止造模后，肝羟脯氨酸含量、血清HA明显降低（P〈0．01），但仍显著高于正常对照组（P〈0．01）。纤维化形成率为100％，纤维化持续时间120d以上。结论 此造模方法简单，肝纤维化成功率高，维持时间长，可用于抗纤维化药物的研究。     

实验性肝纤维化大鼠层粘连蛋白含量的变化

用四氯化碳诱导大鼠肝脏纤维化，采用酶联免疫吸附法（ＥＬＩＳＡ）检测大鼠血清和肝组织匀浆中层粘连蛋白（ＬＮ）含量，并分析ＬＮ与肝功能及肝组织学病理改变之间的关系。结果发现，血清ＬＮ在大鼠肝脏受损伤后早期即出现增加，并随肝组织损害程度的加重而递增，与肝功能改变及病理损伤有一定相关性。肝匀浆中ＬＮ含量在肝损伤晚期才升高，与肝功能指标没有相关性，提示，血清ＬＮ含量改变是反映肝脏损伤的敏感指标，可用于动态观     

透明质酸及其相关蛋白与大鼠肝纤维化的关系

目的:探讨血清透明质酸(HA)及其相关蛋白(SHAP-HA)水平与肝纤维化间的关系.方法:建立实验性肝纤维化动物模型,检测血清HA、SHAP-HA、谷-丙转氨酶(ALT)、谷-草转氨酶(AST)、白蛋白(ALB),观察大鼠肝组织病理变化.结果:随着肝纤维化程度的加重,血清HA、SHAP-HA表达量均增加,与ALT、AST呈正相关,差异具有统计学意义.结论:实验性肝纤维化过程中HA、SHAP-HA表达水平不仅与肝组织的炎症坏死程度有关,而且与纤维化过程关系密切,HA、SHAP-HA可作为肝纤维化评估指标之一.     

鸭乙型肝炎肝纤维化模型的研究

目的 :用鸭乙型肝炎病毒阳性血清反复攻击建立鸭乙型肝炎肝纤维化动物模型。方法 :用鸭乙型肝炎病毒阳性血清0 1ml/只 (含DHBV颗粒 0 5× 10 9)从胫静脉注射 1日龄樱桃谷鸭 ,每周 1次 ,从每 10周剂量加大为 0 2ml/只 (含DHBV颗粒 2 5× 10 9) ,16周末处死动物 ,观察肝纤维化指标及肝脏组织病理学变化。结果 :用此方法诱发鸭肝纤维化形成率达87 5 % ,与四氯化碳腹腔注射诱发肝纤维化形成率接近 ,肝纤维化指标羟脯氨酸、透明质酸和Ⅲ型前胶原明显增高 ,与肝纤维化程度一致。结论 :用鸭乙型肝炎病毒阳性血清反复攻击复制鸭乙型肝炎肝纤维化模型是成功的     

三氯乙烯药疹样皮炎患者肝功能与肝纤维化指标变化

目的 :探讨三氯乙烯药疹样皮炎患者肝功能损害和肝纤维化标志物的变化。方法 :选择 30名三氯乙烯药疹样皮炎患者及 30名健康者作为对照组 ,采用放射免疫分析 (RIA)检测血清透明质酸 (HA)、层粘连蛋白 (LN)、Ⅲ型前胶原 (PCⅢ )、Ⅳ型胶原 (Ⅳ .C)及甘胆酸 (CG)水平 ;化学法显色测定血清单胺氧化酶 (MAO) ;全自动生化仪测定转氨酶 (ALT)、转酞酶 (GGT)、总蛋白 (TP)、白蛋白 (Alb)胆汁酸 (TBA)等肝功能变化指标 ;分析各项指标的相关性。结果 :三氯乙烯药疹样皮炎患者各项肝功能指标变化明显 ;肝纤维化指标MAO、HA、PCⅢ、Ⅳ、CG水平明显增高 (P <0 0 1) ,与ALT、GGT、TP、Alb有良好的相关性。结论 :三氯乙烯药疹样皮炎患者早期肝脏损害严重并伴有肝脏纤维化现象。     

肝毒清颗粒对大鼠实验性肝纤维化的防治作用

目的 :观察肝毒清颗粒的抗纤维化作用。方法 :将Wistar雄性大鼠随机分成 6组 ,即正常对照组、模型组、肝毒清大、中、小剂量组和乙肝宁阳性组 ,采用四氯化碳诱导肝纤维化模型。于造模第 2个月始给予治疗药物。实验持续 3月后将大鼠处死取血作肝功检查及取肝组织做病理检查。结果 :肝毒清能降低AST ,升高TP、ALB ,与模型组比较 (P <0 .0 5 ) ;减轻肝脂肪变性、减少纤维组织增生、促进肝细胞再生。结论 :肝毒清对大鼠肝纤维化有明显防治作用     

中药肝复康对实验性肝纤维化大鼠肝功能及血清标志物的影响

目的 :研究肝复康对实验性肝纤维化大鼠肝功能及血清标志物的影响。方法 :采用四氯化碳致大鼠肝纤维化模型 ,经肝复康干预后分别以赖氏法、溴甲酚绿比色法测定血清转氨酶及白蛋白、球蛋白活性 ,放免法测定血清透明质酸 (HA)、层粘连蛋白 (LN)、Ⅳ型胶原 (C -Ⅳ )、Ⅲ型前胶原 (PC -Ⅲ )。结果 :肝复康治疗后血清转氨酶、球蛋白活性降低 ,白蛋白活性升高 ,血清HA、LN、C -Ⅳ、PC -Ⅲ均有明显降低 ,与模型组比较有显著差异 (P <0 .0 1 )。结论 :肝复康对实验性肝纤维化有明确的治疗作用 ,降低肝纤维化程度 ,同时有效减轻肝细胞损伤 ,改善肝功能。     

实验性肝纤维化大鼠胶原网络构筑的扫描电镜观察

目的 探讨肝纤维化大鼠胶原网络的三维形态构筑的变化。方法 采用健康雄性Wislar大鼠,随机分为对照组和肝纤维化组,后者采用60％的四氯化碳植物油皮下注射,造成肝纤维化大鼠模型,分别于实验的第10周和12周末取材,作为10周和12周肝纤维化组,行天狼猩红显色,光镜下观察和氢氧化钠浸渍,扫描电镜下观察胶原网络的构筑。结果 扫描电镜下,随着肝纤维化程度的加重,增生的胶原纤维交织成网,分隔包裹正常肝组织,形成大小不等圆形或椭圆型肝组织团块,近似于“蜂窝状”囊性的假小叶结构;相邻假小叶之间的胶原纤维沉积明显,并与包裹门管区和血管的纤维鞘相延续为间隔。与对照组相比,10周肝纤维化组形成了尚不完整的假小叶囊,12周组的小叶囊致密完整。结论 肝纤维化时胶原纤维超微构筑发生了改变,增生的胶原网络主要由粗、细纤维构成。     

当归对CC1_4所致肝损伤大鼠的抗肝纤维化作用

将１２０只ＳＤ大鼠随机分为５组，在腹腔注射ＣＣ１＿４的同时，于不同实验阶段投予当归注射液。１２周后，取肝脏作ＶＧ和ＨＥ染色，以观察肝纤维化程度及肝内胶原含量的变化。结果表明，早、中期投予当归注射液组，其肝纤维化程度显著轻于后期投药组，肝内胶原含量亦显著少于后者。提示当归可显著减轻肝纤维化程度，似不能促进己沉积的胶原降解。     

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。     

混合物脑肽干预对肝纤维化大鼠模型的影响

背景：混合物脑肽是从动物脑中提取的多肽混合物,含有神经生长因子成分。近年研究发现,神经生长因子与肝损伤密切相关。 目的：使用自拟混合物脑肽观察对大鼠肝纤维化的影响。 方法：利用四氯化碳诱导形成SD大鼠肝纤维化模型。将大鼠随机分成纤维化模型组、脑肽干预组和对照组。4,8周采集标本。通过活体解剖取门静脉血清采用全自动生化分析仪检测肝功能丙氨酸氨基转移酶、天冬氨酸氨基转移酶、总胆红素、白蛋白;采用放射免疫法检测血清肝纤维化指标透明质酸、层粘连蛋白、Ⅲ型前胶原;常规苏木精-伊红和网状纤维染色检测肝组织标本。 结果与结论：大鼠肝纤维化模型成功建立。脑肽干预组纤维化程度较纤维化模型组显著减轻,但肝组织炎症和肝细胞脂肪变性反而较纤维化模型组更显著。初步判断脑肽能够阻断四氯化碳诱导的肝纤维化,可能与神经生长因子有关。     

Micro RNA与肝病及肝纤维化相关研究

Micro RNA(miRNA)是转录后水平的细胞调控因子.在细胞核内,miRNA基因经由RNA聚合酶Ⅱ介导转录出含多聚腺苷酸尾(AAAAA)和帽子结构特征性标志的primiRNA,后者在核酸酶Drosha的作用下被切割成70个具有茎环结构的miRNA前体(pre-miRNA),随后由转运蛋白exportin-5转运出核,再由胞质中Dicer蛋白切割为成熟的miRNA.miRNA在RNA沉默复合物(RNA-induced silencing complex,RISC)的作用下可与多个互补mRNA结合,从而降解该mRNA或抑制其翻译.目前已在人细胞中发现1048种miRNA,参与调控全基因组中20%～30%基因的表达(MiRBase http://www.mirbase.org/).     

二乙基亚硝胺诱导大鼠肝纤维化模型血清N-糖组表达分析

糖蛋白糖链具有广泛的生物学意义,异常糖基化状态与疾病的发生发展密切相关。肝纤维化易向肝硬化和肝癌发展,早期肝纤维化具有可逆转性,所以早期诊断尤为重要。本研究通过二乙基亚硝胺(DENA)诱导的大鼠肝纤维化模型,验证N-糖标志物在肝纤维化发生发展中的变化过程。用DENA腹腔注射的方法诱导大鼠肝脏纤维化,用DSA-FACE的方法检测血清及血清中纯化的免疫球蛋白的N-糖组图谱。研究发现,peak1,peak2,peakR5a和peakR5b随着肝纤维化程度的加深而升高,而peak5和peak8则降低。Peak2主要来源于免疫球蛋白,可能与肝纤维化过程中的炎症状态密切相关,而大鼠特异性的N-糖peakR5a和peakR5b则主要来源于肝脏,可以评估和检测大鼠肝脏的纤维化状态,从而验证了N-糖标志物作为肝病诊断标志物的可靠性和潜在价值,为进一步筛选肝病的早期诊断指标打下了基础。     

Establishment of a liver fibrosis model in cynomolgus monkeys

Hepatic fibrosis, resulted from hepatoxicity and other diseases such as diabetes, is an important pathological characteristic of chronic liver diseases. Establishment of hepatic fibrosis animal models is of great importance and a prerequisite for human clinical studies. The common models for liver fibrosis are often established in lower small animals such as rats, but non-human primates are a much better model for human diseases because of the physiological similarity with humans. In this study, we investigated the method to induce liver fibrosis in cynomolgus monkeys using carbon tetrachloride (CCl₄) and to establish a model that more closely mimics human liver fibrosis. We successfully established the liver fibrosis model in 15 of the 20 cynomolgus monkeys (success rate 75%), by CCl₄ administration at a dose of 1.0 mL/kg (400 mL/L) twice a week. Liver biopsy showed that liver fibrosis progressed with time and gradually advanced into early-stage cirrhosis in 10 of the 15 established models at 16 weeks. Our study provides a better research platform for the prevention and treatment of chronic liver diseases.     

美洲大蠊提取物黏糖氨酸对肝纤维化模型大鼠的影响及机制

BACKGROUND:Hepatic fibrosis in chronic liver disease can be reversed. Studies have shown that Periplaneta americana extract has anti-fibrosis effect, and has protective effect on the experimental hepatic fibrosis rats. OBJECTIVE:To observe the effect of Periplaneta americana extract sticky sugar amino acid on hepatic fibrosis, and to primarily explore the mechanism of sticky sugar amino acid against hepatic fibrosis. METHODS:Rat models of immune hepatic fibrosis were induced by pig serum and intragastrically administered 0.5, 0.25, 0.10 g/kg sticky sugar amino acid. Four indexes of hepatic fibrosis were detected by radioimmunoassay. Immunohistochemical staining was used to measure transforming growth factor beta 1, tissue inhibitor of metalloproteinase protein expression intensity and positive cell rate, to determine the correlation of different concentrations of sticky sugar amino acid, transformation growth factor beta 1 and tissue inhibitor of metalloproteinase.  RESULTS AND CONCLUSION: (1) The Periplaneta americana extract sticky sugar amino acid reduced the levels of laminin, type III procollagen, type IV collagen and hyaluronidase (P < 0.01) and reduced the expression of transformation growth factor beta 1 and tissue inhibitor of metalloproteinase 1 in liver tissue (P < 0.01). (2) Sticky sugar amino acid concentration and transformation growth factor beta 1 and tissue inhibitor of metalloproteinase 1 protein expression showed a significant negative correlation (| r | > 0.9). (3) Results confirmed that the Periplaneta americana extract sticky sugar amino acid can change the reversal of hepatic fibrosis. Its mechanism of action is associated with expression of transforming growth factor beta 1 and tissue inhibitor of metalloproteinase 1 inhibited by sticky sugar amino acids.     

Bone marrow multipotent mesenchymal stromal cells do not reduce fibrosis or improve function in a rat model of severe chronic liver injury

The objective of our study was to evaluate the therapeutic potential of bone marrow mesenchymal stromal cells (MSC) in a rat model of severe chronic liver injury. Fourteen female Wistar rats were fed exclusively an alcoholic liquid diet and received intraperitoneal injections of carbon tetrachloride every other day during 15 weeks. After this period, eight animals (MSC group) had 1 x 10(7) cells injected into the portal vein while six animals (placebo group) received vehicle. Blood analysis was performed to evaluate alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin before cell therapy and 1 and 2 months after cell or placebo infusion. Fibrosis was evaluated before and 1 month after cell or placebo injection by liver biopsies. Two months after cell delivery, animals were sacrificed and histological analysis of the livers was performed. Fibrosis was quantified by histomorphometry. Biopsies obtained before cell infusion showed intense collagen deposition and septa interconnecting regenerative nodules. One month after cell injection, this result was unaltered and differences in fibrosis quantification were not found between MSC and placebo groups. ALT and AST returned to normal values 2 weeks after cell or placebo infusion, without significant differences between experimental groups. Two months after cell or placebo injection, albumin had also returned to normal values and histological results were maintained, again without differences between MSC and placebo groups. Therefore, under our experimental conditions, MSC were unable to reduce fibrosis or improve liver function in a rat model of severe chronic liver injury.     

解耦联蛋白2对大鼠肝纤维化形成中星形细胞活化的影响

目的探讨解耦联蛋白2(UCP2)在肝纤维化形成过程中的作用及其发生机制。方法体内实验采用四氯化碳(CCl4)诱导肝纤维化模型,取肝脏观察病理变化,用Western blotting、免疫组织化学和Real-time PCR等方法检测UCP2和p38丝裂素活化蛋白激酶(p38MAPK)的表达水平;体外实验采用UCP2特异性抑制剂京尼平和CCl4刺激星形细胞,检测UCP2和p38MAPK相关蛋白的表达情况。结果与正常组相比,模型组大鼠肝脏α-平滑肌肌动蛋白(α-SMA)和UCP2表达增高(P0.05,n=10);加入CCl4刺激细胞后,星形细胞α-SMA表达增加,p38MAPK及其磷酸化水平增高(P0.05,n=6);而在加入京尼平后,α-SMA表达增加,p38 MAPK及其磷酸化水平明显降低(P0.05,n=6)。结论 UCP2参与了肝纤维化的发生,可能促进了星形细胞的活化及增殖过程。     

Polycystic kidney rat is a novel animal model of Caroli's disease associated with congenital hepatic fibrosis

Caroli's disease (congenital intrahepatic biliary dilatation) associated with congenital hepatic fibrosis is an autosomal recessive polycystic kidney disease. Recently, the polycystic kidney (PCK) rat, a spontaneous mutant derived from a colony of CRJ:CD rats with polycystic lesions in the liver and an autosomal recessive mode of inheritance, was reported. In the present study, the pathology of the hepatobiliary system and the biliary cell-kinetics were evaluated in fetuses (day 18 to 21 of gestation) and neonates and adults (1 day to 4 months after delivery) of PCK rats. CRJ:CD rats were used as a control. Multiple segmental and saccular dilatations of intrahepatic bile ducts were first observed in fetuses at 19 days of gestation. The dilatation spread throughout the liver and the degree of dilatation increased with aging. Gross and histological features characterizing ductal plate malformation were common in the intrahepatic bile ducts. Overgrowth of portal connective tissue was evident and progressive after delivery. These features were very similar to those of Caroli's disease with congenital hepatic fibrosis. Proliferative activity in the biliary epithelial cells was greater in PCK rats than controls during the development. In contrast, the biliary epithelial apoptosis was less extensive in PCK rats than the controls until 1 week after delivery, but greater after 3 weeks, suggesting that the remodeling defect in immature bile ducts associated with the imbalance of cell kinetics plays a role in the occurrence of intrahepatic biliary anomalies in PCK rats. The PCK rat could be a useful and promising animal model of Caroli's disease with congenital hepatic fibrosis.