四氯化碳肝纤维化大鼠肝组织差异蛋白质组的动态变化

目的 探索肝纤维化形成过程中肝组织蛋白质组的变化,部分解析肝纤维化发生、发展的病理生理机制. 方法 采用四氯化碳(CCl4)诱导大鼠肝纤维化模型,应用双向凝胶电泳技术分离大鼠肝组织总蛋白质,经考马斯亮蓝染色、图像分析、识别差异表达的蛋白质点,应用基质辅助激光解吸电离飞行时间串联质谱及数据库查询鉴定差异蛋白质点.采用Western blot、免疫组织化学方法对细胞角蛋白(CK)8、CK18进行蛋白质表达水平的验证.计量资料采用单因素方差分析中的LSD法或非参数检验中的H检验进行统计学分析. 结果 大鼠造模后,随时间的推移,肝纤维化评分逐渐增加(3周组＜6周组＜9周组),建立了正常大鼠和大鼠CCl4肝纤维化形成过程3、6、9周肝组织双向凝胶电泳图谱,质谱鉴定了44种差异蛋白质;采用Western blot、免疫组织化学方法对CK8、CK18的验证结果与双向凝胶电泳结果基本一致.正常组及3、6、9周模型组CK8/CK18蛋白相对表达量分别为:0.113±0.005/0.170±0.030、0.473±0.046/0.530±0.070、0.682±0.087/0.780±0.080、0.837±0.096/1.390±0.130,各组间差异均有统计学意义(F值分别为196.085/74.088、13.870/16.115、75.800/75.900,P值均＜0.01). 结论 CCl4大鼠肝纤维化形成与发展过程中差异表达蛋白质的功能主要涉及细胞生长、发育与分化,细胞增殖与凋亡,血管新生或重构,氧化应激,物质代谢及转运,信号转导等.     

复方鳖甲软肝片治疗免疫性肝纤维化大鼠肝细胞质膜蛋白质组分的变化

目的研究复方鳖甲软肝片治疗肝纤维化大鼠肝细胞质膜蛋白质组分的变化,以寻找与肝纤维化逆转相关的质膜蛋白质。方法以猪血清腹腔注射大鼠8周制备肝纤维化模型,再以复方鳖甲软肝片灌胃治疗8周。采用蔗糖密度梯度离心获得肝组织匀浆和质膜组分,采用免疫印迹法检测质膜的纯度,分析差异蛋白质组学变化。结果病理学检查发现实验8周后,大鼠肝脏发生中度肝纤维化;经鳖甲软肝片治疗8周后肝纤维化程度减轻,基本恢复到正常水平;免疫印迹检测显示质膜组分得到了较好的富集,其中线粒体等其他细胞器蛋白减少;通过差异蛋白质组学分析发现,治疗组中有22个3倍以上差异蛋白质点,准确鉴定了9个蛋白质,通过免疫印迹验证发现了Ⅱ型细胞支架蛋白8和膜联蛋白A2的差异表达。结论鳖甲软肝片治疗后,大鼠肝纤维化发生了逆转,并伴随一些质膜蛋白质的表达变化。     

肝纤维化患者血清差异蛋白质的筛选

目的: 对比分析肝纤维化患者和健康人血清中差异表达的蛋白质, 筛选与肝纤维化发生密切相关的血清蛋白质.方法: 肝纤维化患者和健康人空腹血清标本各6例分别等量混合成2个样本, 去除血清中的白蛋白和IgG. 利用双向凝胶电泳(2-DE)分离肝纤维化血清和健康人血清总蛋白质后, 硝酸银染色, 经图像分析筛选差异表达的蛋白点. 应用基质辅助激光解吸电离飞行时间质谱(MALDITOF-MS)鉴定差异表达的蛋白点, 并用ELISA法对其中部分差异蛋白的表达水平进行验证.结果: 筛选出2组间的差异蛋白点共517个, 对其中表达量差异3倍以上的24个蛋白点进行MALDI-TOF-MS分析, 鉴定出包括触珠蛋白、载脂蛋白A-Ⅳ、转铁蛋白、T细胞受体b链、血清白蛋白及血清白蛋白前体等8个蛋白质, 其中3个蛋白质在肝纤维化血清中表达上调, 5个蛋白质在肝纤维化血清中表达下调. 与健康人血清比较, ELISA法检测肝纤维化血清中触珠蛋白、载脂蛋白A-Ⅳ表达均下调, 结果与双向凝胶电泳一致.结论: 肝纤维化血清与健康人血清的蛋白质表达谱表现出一定差异, 这些差异表达的血清蛋白质有望成为肝纤维化诊断与判定预后的血清标志物.     

CCl4大鼠肝纤维化形成与消减过程中肝组织蛋白质组的动态变化及扶正化瘀方的干预作用

目的:通过大鼠纤维化肝组织高亲水性蛋白质分子差异展示及扶正化瘀方干预作用,寻找与扶正化瘀方治疗肝纤维化相关的蛋白质分子.方法:通过皮下注射大鼠CCl4,分别持续4、8、12周,对大鼠肝纤维化过程进行动态观察,并在不同时期进行扶正化瘀方干预,治疗从第12周始至16周止.采用蛋白质分步抽提方法,提取肝组织蛋白质,分别进行二维聚丙烯酰胺凝胶电泳(2-DE)分离、凝胶银染,显示各组蛋白质分子的差异表达的蛋白质分子.结果:在各组银染凝胶图谱上,可辨识的蛋白质斑点达1 000～1 400个左右,蛋白质表达中除有肝组织看家蛋白质,模型组也有特异蛋白质表达,且在不同时期蛋白质分子表达也有差异,特异表达蛋白质分子为100至300多个不等,在不同阶段表达是不相同的,经质谱鉴定确定了有几类功能不同的蛋白质分子.结论:扶正化瘀方抗肝纤维化作用机制是多方面的,其干预作用与多种蛋白质分子相关.     

不同肝纤维化程度慢性乙型肝炎患者的血浆蛋白质组学分析

目的 探索与肝纤维化程度相关的新血浆标志物,为开发有临床应用价值的肝纤维化无创诊断提供新型检测分子. 方法 收集慢性乙型肝炎患者血浆,并根据肝活组织病理学诊断肝纤维化的分期进行分组,包括S0～1组40例,S2 ～3组20例和S4组20例.血浆样品去除血浆中的白蛋白和免疫球蛋白G后,通过二维凝胶电泳进行分离,采用ImageMaster软件分析获得的电泳图谱,找到与疗效相关的差异蛋白质.差异蛋白质点经过胰酶酶切后,通过在线纳升级反向液相色谱串联电喷雾离子阱质谱分析进行鉴定.结果 通过比较不同组血浆蛋白质的二维凝胶电泳图谱,共发现了14个在S0～1组、S2～3组和S4组血浆样品中表达差异的蛋白质点.液相色谱串联质谱分析鉴定差异蛋白质点,包括人纤维蛋白原Y链、接联球蛋白、补体蛋白C3、免疫球蛋白κ链C区和载脂蛋白A-I.结论 通过对不同肝纤维化程度的慢性乙型肝炎患者血浆进行蛋白质组学分析,发现和鉴定了5种在乙型肝炎患者血浆中表达水平与肝纤维化进程相关的差异蛋白质,这些蛋白质可能是评价患者肝纤维化程度的潜在生物标志物.     

扶正化瘀方对肝纤维化大鼠不同病理阶段肝组织蛋白质组变化的影响

目的探讨四氯化碳（CCl4）大鼠肝纤维化形成及消减不同时期肝组织蛋白质组的动态变化及扶正化瘀方的干预作用。方法40％CCl4-橄榄油溶液皮下注射法制备CCl4大鼠肝纤维化模型，治疗组给予扶正化瘀方灌胃，在第4、8、12和第16周末分批处死大鼠，观察各组大鼠肝组织病理学、羟脯氨酸（Hyp）含量变化，并提取肝组织蛋白质进行二维电泳，运用PDQUEST 2-DE软件对蛋白质图谱进行分析，以MALDI—TOF—MS鉴定差异表达的蛋白质。结果（1）二维电泳结果：模型大鼠特有蛋白质和表达量差异的蛋白质从第4周始出现，第12周达顶峰，（2）质谱鉴定的蛋白质多数在第4周出现，四个时间段均出现的有10个，其中有8个是正常组所没有的，（3）扶正化瘀方对表达差异的蛋白质调节表现在：第4、8周对上调的蛋白质降低作用显著，在第8、12、16周对下调的蛋白质的升高作用较显著，模型大鼠特有的蛋白质经扶正化瘀方干预后，通过降解、下调表达量等形式，趋向正常表达。结论（1）蛋白质表达改变是肝纤维化整体病变的物质基础。表达异常的蛋白质涉及机体的物质代谢、神经内分泌、细胞增殖凋亡等系统，（2）扶正化瘀方在抗细胞增生、促进正常蛋白质合成等方面有综合优势，通过对多个蛋白质、多途径的综合调节达到抗肝纤维化的效果。     

Cyp4 A2,Apoa1,ANXA2及DPYD为金雀异黄酮抗肝纤维化大鼠的关键蛋白

目的:分析金雀异黄酮作用肝纤维化大鼠后,肝组织中蛋白质的差异表达,以探讨药物作用的关键蛋白.方法:将SD大鼠分为对照组、模型组和金雀异黄酮组,除对照组外,其余各组灌胃50%CCl4溶液连续15 wk,于第9周开始以金雀异黄酮组进行给药治疗,连续6 wk之后,取大鼠肝组织,提取蛋白质,以同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,i TRAQ)技术标记蛋白,经质谱定量、鉴定蛋白质,采用生物信息学分析蛋白质的功能;以Western blot对有代表性的药物作用关键蛋白细胞色素C 4A2(Cyp4 A2)和载脂蛋白A1(apolipoprotein A-1,Apoa1)进行验证.结果:质谱共鉴定到585种蛋白质,其中差异蛋白为63个,含上调蛋白32个,下调蛋白31个.对这些差异表达蛋白进行功能分析,发现参与信号传导、分子催化、分子绑定等过程,差异表达蛋白之间存在紧密的相互作用.Western blot验证显示,与模型组相比,蛋白质相互作用的关键节点蛋白Cyp4 A2的表达明显降低,而Apoa1的表达明显增加.结论:金雀异黄酮能通过调节肝组织中的多种蛋白质表达从而达到抗肝纤维化的效果,其中对Cyp4 A2、Apoa1、膜联蛋白A2、二氢嘧啶脱氢酶等蛋白的调节可能是其抗肝纤维化作用的关键.     

大鼠酒精性肝纤维化肝非实质细胞蛋白质组学研究

目的本文研究酒精对肝非实质细胞蛋白质表达的影响,以探讨酒精性肝纤维化的发病机制。方法大鼠酒精灌胃导致其发生肝纤维化。采用James染色法检测大鼠肝脏的病理学变化,通过Percoll密度梯度离心富集肝非实质细胞,再通过二维凝胶电泳(2DE)分离非实质细胞的蛋白质,经考马斯亮蓝(G250)染色,采用液相色谱串联质谱鉴定差异表达的蛋白质,并对部分差异蛋白质采用实时定量RT-PCR和免疫印迹的方法进行验证。对于2DE胶上的蛋白质点,采用两样本t检验的方法,对于RT-PCR分析,采用Mann-Whitney U检验进行统计分析。结果建立了酒精性肝纤维化大鼠模型,通过Percoll密度梯度离心纯化的非实质细胞中淋巴细胞、Kupffer细胞和内皮细胞分别富集了1.5、3.2和3.7倍。采用二维凝胶电泳法检测到了800多个蛋白质点,检测到具有2倍以上的差异蛋白质有26个,采用LC-MS法鉴定了21个非冗余蛋白质,对其中7个蛋白质的RT-PCR分析发现:ANXA3、CES3、ATPA和NDUFV2的mRNA水平和蛋白质组研究结果一致。结论本研究鉴定了一批与酒精性肝纤维化相关的蛋白质,可能为了解酒精性肝纤维化发病机制提供一些新线索。     

DJ-1蛋白在晚发型阿尔茨海默病蛋白组学的表达

目的 从蛋白质水平初步探讨晚发型阿尔茨海默病发病机制,寻找并鉴定与之有关的生物标志物.方法 选择经病理证实的8例晚发型阿尔茨海默病患者及5例正常老年人颞叶脑皮质为研究对象.分别进行双向凝胶电泳后采用基质辅助激光解析-电离-飞行时间质谱及电喷雾电离串联质谱法进行质谱分析,搜索数据库鉴定蛋白质.结果 分别获得两组脑组织双向凝胶电泳蛋白质组表达图谱,DJ-1蛋白在疾病组明显上调.结论 DJ-1蛋白可能成为具有临床诊断意义的晚发型阿尔茨海默病潜在标志物,从而帮助开发靶向目的 蛋白质的新型抗神经退化药物.     

乙型肝炎肝纤维化组织双向电泳技术的优化

目的:建立并优化乙型肝炎肝纤维化组织蛋白质组研究所需的双向凝胶电泳技术,提高其分辨率和重复性.方法:外科手术获取乙型肝炎肝纤维化组织,提取肝组织总蛋白.利用固相pH梯度双向凝胶电泳技术分离总蛋白,考马斯亮蓝染色后分析凝胶图像.对2-DE中的关键因素与环节,如样本处理、上样量、电泳参数等各步条件进行了一系列优化.结果:优化条件后的乙型肝炎肝纤维化组织双向凝胶电泳图谱水平拖尾减少、斑点分辨率改善、斑点检出数量增加(246±33→729±83).结论:该双向凝胶电泳技术可应用于乙型肝炎肝纤维化组织的蛋白质组学研究.     

Study of heteroserum-induced rat liver fibrosis model and its mechanism

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Experimental models of hepatic fibrosis: a review

No experimental model reproduces exactly human liver fibrosis by etiology. Nonetheless, each of the models reviewed in this article has served to enhance our understanding of pathogenetic mechanisms of liver fibrosis. There have been important common findings derived from several different models. The best example is the role of Ito cells in liver fibrogenesis. Involvement of Ito cells was consistently seen in the experimental models regardless of whether the fibrogenic stimulus was nutritional, hepatotoxic, or immunologic. Cellular and molecular mechanisms of Ito cell activation have begun to be explored in different models. Another example is the role of TGF beta in liver fibrogenesis. In both murine schistosomiasis model and Tsukamoto-French rat model, TGF beta was shown to be closely associated with fibrogenesis. With both in vitro and in vivo experimental approaches using cellular and molecular techniques, the experimental model of liver fibrosis will continue to provide data on the pathogenetic mechanisms of liver fibrogenesis. Future genetic and molecular approaches may allow development animal models with liver fibrosis that is inducible and genetically similarity to that of man.     

Study of heteroseruminduced rat liver fibrosis model and its mechanism

AIM: To investigate the morphological changes in the process of heteroserum induced rat liver fibrosis and the mechanism of fibrogenesis of this model.METHODS: A model of heteroserum-induced rat liver fibrosis was established by intraperitoneal injection of porcine serum. In addition to the observation of the morphological changes of this model, the infiltration of eosinophils and mast cells were measured quantitatively and the deposition of IgG and complement C₃ was detected by immunofluorescence.RESULTS: The rat liver fibrosis was induced successfully at the end of the 8th week after the injection of heteroserum. Besides the increase of hepatic stellate cells (HSC) during the process of liver fibrosis, proliferation and activation of primary mesenchyma cells (PMCs) were also found. In the early stage, the infiltration of eosinophils and mast cells was significantly increased and the deposition of IgG and complement C₃ was positive in the portal tracts and septa, while gradually reduced after the injection was stopped.CONCLUSIONS: This model is suitable for the research on liver fibrogenesis; the pathogenesis of this model may be related with the allergen-induced late phasereaction (LPR) caused by the injection of heteroserum, and the HSCs and the PMCs are important sources of ECM-producing cells.     

腱蛋白在四氯化碳大鼠肝纤维化形成过程中的动态表达

目的：探讨腱蛋白（Tenascin,Tn)在肝纤维化形成中的作用。方法：以CCl4诱发大鼠肝损伤期（4周），肝纤维化早期（8周）和肝纤维化晚期（12周）模型；采用免疫组织化学及核酸原位分子杂交技术对大鼠肝组织中Tn mRNA及其蛋白质表达进行观察，并进行计算机图像分析。结果：正常肝组织有少量Tn分布；肝损伤期及肝纤维化早期，Tn的mRNA及蛋白质表达均显著增高。肝纤维化晚期，Tn的mRNA及蛋白质表达相对减少，核酸原位杂交提示肝间质细胞是Tn的主要合成细胞。结论：Tn是肝内重要的细胞外基质成分，参与了肝纤维化的早期形成。     

JTE-522, a cyclooxygenase-2 inhibitor,is an effective chemopreventive agent against rat experimental liver fibrosis1

BACKGROUND & AIMS: The aim of this study was to assess the effects of cyclooxygenase (COX)-2 inhibition on rat experimental liver fibrogenesis. METHODS: We investigated the inhibitory effects of a selective COX-2 inhibitor, JTE-522, on liver fibrosis induced by a choline-deficient, l-amino acid-defined diet (CDAA). Inhibitory effect was also tested in a second model of thioacetamide (TAA)-induced liver fibrosis. RESULTS: CDAA induced liver fibrosis and preneoplastic foci at 12 weeks and cirrhosis at 36 weeks. Hepatocellular carcinoma was noted in 13 of 15 rats (87%). JTE-522 significantly inhibited fibrosis and development of preneoplastic lesions in a dose-dependent manner and completely inhibited generation of cirrhosis and hepatocellular carcinoma at both low and high doses (10 and 30 mg/kg body wt/day, respectively). JTE-522 administrated only from 12 weeks to 36 weeks also prevented cirrhosis and formation of hepatocellular carcinoma. JTE-522 itself did not cause local or systemic gross or histopathologic changes at 36 weeks. Mechanistic studies indicated that the CDAA model displayed up-regulation of several biomarkers, including COX-2, arachidonate metabolite (prostaglandin E(2)), serum aspartate aminotransferase, and c-myc expression. The model also showed an increased proportion of activated hepatic stellate cells, proliferating cell nuclear antigen index, and CD45-positive inflammatory cells in the liver. JTE-522 effectively diminished these changes. JTE-522 exhibited similar antifibrosis effects in the TAA model. CONCLUSIONS: Our results suggest that COX-2 is involved in CDAA- and TAA-induced liver fibrosis. Our data also indicate that JTE-522 is a potent chemopreventive agent of rat liver fibrosis with low toxicity.     

Dendritic cells regulate angiogenesis associated with liver fibrogenesis

During liver fibrogenesis the immune response and angiogenesis process are fine-tuned resulting in activation of hepatic stellate cells that produce an excess of extracellular matrix proteins. Dendritic cells (DC) play a central role modulating the liver immunity and have recently been implicated to favour fibrosis regression; although their ability to influence the development of fibrogenesis is unknown. Therefore, we explored whether the depletion of DC during early stages of liver injury has an impact in the development of fibrogenesis. Using the CD11c.DTR transgenic mice, DC were depleted in two experimental models of fibrosis in vivo. The effect of anti-angiogenic therapy was tested during early stages of liver fibrogenesis. DC depletion accelerates the development of fibrosis and as a consequence, the angiogenesis process is boosted. We observed up-regulation of pro-angiogenic factors together with an enhanced vascular endothelial growth factor (VEGF) bioavailability, mainly evidenced by the decrease of anti-angiogenic VEGF receptor 1 (also known as sFlt-1) levels. Interestingly, fibrogenesis process enhanced the expression of Flt-1 on hepatic DC and administration of sFlt-1 was sufficient to abrogate the acceleration of fibrogenesis upon DC depletion. Thus, DC emerge as novel players during the development of liver fibrosis regulating the angiogenesis process and thereby influencing fibrogenesis.     

Molecular mechanisms of alcohol-induced hepatic fibrosis

Alcohol abuse is a major cause of liver fibrosis and cirrhosis in developed countries. Before alcoholic liver fibrosis becomes evident, the liver undergoes several stages of alcoholic liver disease including steatosis and steatohepatitis. Although the main mechanisms of fibrogenesis are independent of the etiology of liver injury, alcoholic liver fibrosis is distinctively characterized by a pronounced inflammatory response due to elevated gut-derived endotoxin plasma levels, an augmented generation of oxidative stress with pericentral hepatic hypoxia and the formation of cell-toxic and profibrogenic ethanol metabolites (e.g. acetaldehyde or lipid oxidation products). These factors, based on a complex network of cytokine actions, together result in increased hepatocellular damage and activation of hepatic stellate cells, the key cell type of liver fibrogenesis. Although to date removal of the causative agent, i.e. alcohol, still represents the most effective intervention to prevent the manifestation of alcoholic liver disease, sophisticated molecular approaches are underway, aiming to specifically blunt profibrogenic signaling pathways in liver cells or specifically induce cell death in activated hepatic stellate cells to decrease the scarring of the liver.