肝硬化模型比较蛋白质组学研究及扶正化瘀方对血浆蛋白质组影响
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摘要
第一部分肝硬化模型肝组织比较蛋白质组学研究
研究背景与目的：肝硬化是临床常见的慢性进行性肝病，是我国疾病防治的主要任务之一。肝纤维化是肝硬化发展的必经病理过程。蛋白质组学应用各种技术手段从整体角度分析蛋白质组成、表达水平与修饰状态的动态变化，了解蛋白质之间的相互作用与联系，揭示蛋白质功能与细胞生命活动规律。疾病蛋白质组学为探讨疾病的发病机制、预防、早期诊断和治疗提供重要理论基础。本实验构建CCl_4诱导大鼠肝硬化模型，建立肝组织蛋白质组研究的技术平台；掌握进行大鼠肝组织双向凝胶电泳所需要的各种条件；了解CCl_4诱导大鼠肝硬化模型肝组织蛋白质组变化，为进一步研究肝纤维化、肝硬化发病机制奠定基础。
方法：将雄性S-D大鼠随机分成3组：正常对照组(n=6)给予橄榄油腹腔注射；肝硬化模型组(n=10)用CCl_4(CCl_4／橄榄油：v／v=1／1)按1ml／kg腹腔注射，每周2次，共8周诱导肝硬化模型。病理组织学观察肝纤维化严重程度并评分，用Masson三色染色法进行胶原染色。计数肝窦内α-SMA阳性细胞数观察肝星状细胞的活化情况。对肝组织进行双向凝胶电泳，应用PDQUST图像分析软件将肝硬化模型组与对照组肝组织2-DE胶图进行匹配，不同胶匹配点相对含量差异2倍及其以上的点为差异蛋白质点。对差异蛋白质点进行胶内消化、MALDI-TOF-TOF-MS质谱仪鉴定和数据库搜索确定差异蛋白质。用免疫印迹法验证双向凝胶电泳质谱技术鉴定的蛋白质表达改变。
结果：肝硬化模型组肝组织病理均提示典型纤维间隔和假小叶形成，α-SMA阳性细胞表达明显增加。肝组织双向电泳凝胶重复性满意，显示的蛋白斑点在分布、背景等方面基本一致。通过PDQUST软件分析的差异蛋白质点经胶内消化、质谱鉴定、蛋白质数据库搜索有33个点得到鉴定，分别属于28种蛋白质。在这些差异蛋白质中，有5种蛋白质仅在肝硬化模型组中出现；2种蛋白质仅在正常组出现；12种蛋白质在肝硬化模型组上调，9种蛋白质下调。这些蛋白质按其功能特性可分为与细胞增殖、能量代谢相关酶类、物质代谢和转运相关蛋白、细胞骨架和运动相关蛋白、氧化应激、内质网应激和热休克相关蛋白质。免疫印迹法进一步证实了蛋白质组技术显示的alpha crystallin B chain变化趋势。
结论：成功构建肝硬化大鼠动物模型。利用蛋白质组学技术，从整体角度将蛋白质变化综合一起，得到CCl_4诱导肝硬化大鼠肝组织蛋白质组的特征性变化。在CCl_4诱导大鼠肝硬化形成过程中，肝组织中差异表达的蛋白质可能通过影响细胞增殖、能量代谢、物质转运代谢、细胞骨架形成和运动、应激反应等参与、反映肝硬化的发生发展过程。
第二部分肝硬化模型血浆比较蛋白质组学研究
研究背景与目的：许多疾病可导致血浆中多种蛋白质改变，这种特征性变化对疾病诊断和疗效监测具有重要意义。迄今为止人类仅对少数血浆蛋白作为临床常规监测用于某些疾病的诊断。血浆蛋白质组的发展拓宽了对血浆蛋白质的认识。全面而系统地认识肝纤维化和肝硬化时血浆蛋白改变情况，将有助于肝纤维化、肝硬化的发病机制、诊断和药效研究。本实验将初步建立血浆蛋白质组技术平台；掌握去除大鼠血浆白蛋白和球蛋白的方法以及进行血浆双向凝胶电泳所需要的各种条件；了解CCl_4诱导大鼠肝硬化疾病模型的血浆蛋白质组变化，为进一步研究肝硬化的发病机制和诊断奠定基础。
方法：用CCl_4制备肝硬化动物模型，单纯橄榄油腹腔注射组作为对照组。造模8周后抽取血液，同组间血浆混匀，去除血浆中白蛋白和球蛋白。进行双向凝胶电泳，应用PDQUST图像分析软件对各种条件下除血浆白蛋白和球蛋白的效果进行评价。将肝硬化模型组与对照组血浆2-DE胶图进行匹配，不同胶匹配点相对含量的差异2倍及其以上的点为差异蛋白质点。对差异蛋白质点进行胶内消化、MALDI-TOF-TOF-MS质谱仪鉴定和数据库搜索鉴定差异蛋白质。用免疫印迹法验证双向凝胶电泳质谱技术所鉴定的蛋白质表达改变。
结果：使用Albumin／IgG Removal Kit可去除血浆中大部分白蛋白和球蛋白。去除后，双向凝胶电泳显示的蛋白质斑点由处理前的260±15个增加到598±42个。胶重复性满意，显示蛋白斑点在分布、背景等方面基本一致。通过PDQUST软件分析的差异蛋白质点经胶内消化、质谱鉴定、蛋白质数据库搜索有20个点得到鉴定，分别属于16种蛋白质：血浆谷胱甘肽过氧化物酶、谷胱甘肽过氧化物酶前体、载脂蛋白A-Ⅳ前体、载脂蛋白E、结合珠蛋白、前白蛋白、血浆视黄醇结合蛋白、α1-抗胰蛋白酶、微管相关调节激酶1、α1-巨球蛋白、补体4、α抑制剂H4重链、转铁蛋白、肝脏再生相关蛋白、波形纤维蛋白和RIKEN cDNA1700025816。免疫印迹法进一步证实了蛋白质组研究显示的结合珠蛋白变化趋势。
结论：去除大鼠血浆中的白蛋白和球蛋白可明显增加双向凝胶电泳蛋白质点的显示，优化了血浆2-DE图谱。本研究建立的血浆蛋白质组研究平台可用于疾病机制的研究。CCl_4诱导大鼠肝硬化模型血浆中细胞防御和免疫相关蛋白质、物质转运蛋白质、细胞骨架和转化调控蛋白、急性时相反应蛋白等在表达量上不同程度地发生了变化，提示这些蛋白质表达改变可能与肝硬化的发病机制有关，反映了肝硬化时血浆蛋白质的整体改变。
第三部分扶正化瘀方干预肝硬化大鼠血浆比较蛋白质组研究
研究背景与目的：运用蛋白质组学方法，分析中药复方对CCl_4诱导肝纤维化、肝硬化大鼠血浆与正常血浆蛋白质组差异表达，以期筛选出与药物作用相关的差异蛋白质，为肝纤维化、肝硬化的药物治疗机制和疗效评价提供依据。目前扶正化瘀方在治疗肝炎后肝硬化及慢性乙型肝炎肝纤维化方面取得良好的临床效果，其抗肝纤维化作用机制是多方面的，具有复方中药的多成分、多环节、多层次、多靶点作用特点，蛋白质组学为从整体水平研究扶正化瘀方开辟了新途径。以CCl_4大鼠肝纤维化模型作为研究对象，探讨肝纤维化过程中血浆蛋白质组表达改变，揭示扶正化瘀方对肝纤维化多途径作用机制及其疗效评价。
方法：将雄性S-D大鼠随机分为三组，正常对照组(n=6)给予橄榄油腹腔注射；肝硬化模型组(n=10)给予CCl_4(CCl_4／橄榄油：v／v=1／1)按1ml／kg腹腔注射，每周2次，共8周诱导肝硬化模型；扶正化瘀方组(n=10)在CCl_4腹腔注射同时给予扶正化瘀方灌胃。8周后通过Masson三色染色法用图像分析仪自动分析肝脏胶原面积。应用双向凝胶电泳技术经银染显色和PDQUST7.3软件分析凝胶图像，对三组中表达均有差异的蛋白质点用基质辅助激光解吸电离飞行时间串连质谱进行鉴定。
结果：扶正化瘀方干预组较模型组肝纤维化程度明显减轻，胶原纤维在肝内沉积减少(8.9％±3.7％vs.12.4％±4.7％，P＜0.05)。实验去除血浆中的高丰度蛋白质可获得重复性较好的血浆蛋白质图谱。共鉴定出10个明显差异表达的蛋白质。在这些蛋白质中，血浆谷胱甘肽过氧化物酶及其前体、前白蛋白、结合珠蛋白、载脂蛋白A-Ⅳ前体、补体4、α抑制剂H4重链和微管相关调节激酶1在肝硬化大鼠血浆中表达减少，扶正化瘀方干预组表达增加，肝脏再生相关蛋白和波形纤维蛋白在肝硬化大鼠血浆中表达增加，扶正化瘀方干预组表达减少。
结论：动物实验证实扶正化瘀方具有抗纤维化、肝硬化的作用。本实验初步探索了药物干预下的血浆蛋白质组研究，从血浆蛋白质整体变化角度阐述了扶正化瘀方抗肝纤维化和肝硬化作用，研究发现扶正化瘀方可能通过促进蛋白质合成、抗氧化、调控细胞增殖和转化等多方面发挥抗纤维化的作用。蛋白质组研究可能为复方中药疗效监测和作用机制研究开辟新途径。
Part one
Hepatic comparative proteomic study of cirrhotic rats induced by CCl_4Background and aims: Cirrhosis is a popular chronic progressive disease in China,and how to cure it is a major task of the Chinese medical specialists. Liver fibrosis isan indispensable pathological status during the development of cirrhosis. Proteomicsis a novel method to determine the constitution of proteins and to analyze the dynamicchanges of the expression and modification of proteins from an integrative level, bywhich the interaction and relationships among proteins are studied and the functionsof proteins and the principles of cellular activities are discovered. Disease proteomicsaims to lay theoretic foundation for studying the mechanisms, prevention,pre-diagnosis and therapy of diseases. In the present study a rat model ofCCl_4-induced cirrhosis was constructed, a platform for proteome of liver tissue wasbuilt up, and the conditions for two-dimensional gel electrophoresis of rat liver tissuewere determined. By analyzing the proteome of the rat model of CCl_4-inducedcirrhosis the study laid a basis for the further research of liver fibrosis, and cirrhosis.Methods: Male S-D rats were randomly divided into three groups: the control group(n=6) was injected with olive oil into abdominal cavity, cirrhotic animal model group(n=10) was treated in the same way with CCl_4(CCl_4/olive oil:v/v=1/1) 1ml/kg twicea week and for 8 weeks. The degree of liver fibrosis and inflammation grade wasobserved using histopathology study, collagen were visualized by Masson-trichrome--staining.α-SMA positive cells in hepatic sinus were counted to observe the activationof hepatic stellate cell. After two-dimensional gel electrophoresis the gels fromcirrhosis group and control group were analyzed by image analysis softwarePDQUST to match same points. Twice or more different content were considereddifferent points. Those points were digested, analyzed with MALDI-TOF-TOF-MSmass spectrograph and searched in protein database. The expression of protein wasidentified by immunoblotting test.
Results: Typical fibrotic septa and pseudo-lobule were observed in cirrhotic animal model. The expression ofα-SMA positive cell increased. The reproducibility wassatisfying, proteins had approximately the same distribution and background. Afterprotein digestion in situ and mass spectrometric analysis, 33 points corresponding to28 proteins were identified. In those different proteins, 5 proteins only appear in livercirrhotic animal model group, 2 proteins only appear in control group. 12 proteinsexpression was promoted and 9 depressed in cirrhotic animal model group. Thoseproteins function in cell proliferation, energy metabolization, substancemetabolizetion and transport of acting enzyme, cytoskeleton and protein movement,oxidation stress, endoplasmic reticulum stress and hot shock stress respectively. Thechange trend of alpha crystalline B chain was further approved by immunoblottingtest.
Conclusions: We successfully established a cirrhotic rat model. The change of thoseproteins was considered integratively, from which we obtained the characteristicchange in the liver tissue of cirrhotic animal induced by CCl_4. On the progress ofcirrhosis, the expression of diverse proteins may participate in the occurring anddeveloping of cirrhosis in the ways of cell proliferation, energy metabolization,substance metabolization, cytoskeleton structure and protein movement, and stressrespond.
Part two
Plasma comparative proteomic study of cirrhotic rats induced by CCl_4Background and aims: Many diseases may affect proteins in plasma. Thecharacteristic changes are important to diagnosing diseases and monitoring curativeeffect. So far, only few plasma proteins are used in clinic as markers of some diseases.With the development of plasma proteome, more plasma proteins are entering ourview. Studying the change of plasma proteins of hepatic fibrosis and cirrhosis helps tounderstand the pathologic process, diagnoses and curative effect. In this research, weestablished a plasma proteomic technologic platform, on which we tried to establishthe methods of eliminating rat plasma albumin and globulin, and to optimize theconditions forplasma two-dimensional gel electrophoresis. It can be served as a basisfor further researches in cirrhosis by studying the change of plasma proteome of CCl_4-induced rat cirrhostic model.
Methods: Cirrhotic rats were induced by CCl_4 injection to abdominal cavity, whileolive oil was administrated in the control group. Eight weeks after injection, bloodwas collected and plasma from the same group was mixed. Albumin andimmunoglobulin were wiped off. After two-dimensional gel electrophoresis, gel mapswere analyzed with PDQUST. Points from model group and control group werematched, and twice or more different content were considered different points. Thosepoints were digested, analyzed with MALDI-TOF-TOF-MS mass spectrograph andsearched from protein database. The expression of protein was identified byimmunoblotting test.
Results: Albumin/IgG Removal Kit can wipe off most of albumin andimmunoglobulin from plasma. After the wiping, the number of gel map protein pointsincreases from 260±15 to 598±42. The repeatability was satisfying, and proteins hadapproximately same distribution and background. After protein digestion in situ andmass spectrometric analysis, 20 points are identified which belongs to 16 kinds ofproteins: plasma glutathione peroxidase, glutathione peroxidase precursor,apolipoprotein A-Ⅳprecursor, apolipoprotein E, haptoglobin, prealbumin,retinol-binding protein, alpha-1-antitrypsin, serine/threonine-protein kinase MARK1,alpha-1-Macroglobulin, complement component 4, inter-alpha inhibitor H4 heavychain, transferring, liver regeneration-related protein LRRG03, vimentin, RIKENcDNA1700025B16. The change trend of haptoglobin was further approved byimmunoblotting test.
Conclusions: Wiping off albumin and immunoglobulin from plasma can increase theprotein points display obviously, and optimize the plasma 2-DE map. The platformestablished in our study can be used in studying disease mechanism. In the liver ofcirrhotic rat model induced by CCl_4, those protein participate in cell defense andimmunity, protein transport and cytoskeleton and transformation, and acute phasereactive proteins. This hint the change of protein may be induced by the pathogenesis ofcirrhosis, those protein may take part in diverse ways during the occurring anddeveloping of cirrhosis.
Part three
The effect of Fuzheng Huayu Decoction on plasma proteome of cirrhotic rats Background and aims: Using proteomics we studied the role of Chinese traditionalmedicine compound on the differential expression of proteins in liver fibrosis andcirrhosis induced by CCl_4. The aim is to find the proteins involved in the certainphysiological and pathological progress, and to lay foundation for the early diagnoseand therapy for liver fibrosis and cirrhosis. Presently, Chinese traditional medicine hasshown obviously advantage in treatment of liver fibrosis. Fuzheng Huayu Decoctionhas nice clinic result in treating post-hepatitis cirrhosis and chronic hepatitis B liverfibrosis. The antifibrotic mechanism is various, and the compound works in amulti-component, multi-step, multi-arrangement, multi-target way. Proteomics opensup a new way in researching Fuzheng Huayu Decoction from an integrative level. Westudied the change of plasma proteome in the progress of cirrhosis in rat modelinduced by CCl_4, and tried to find how the multi-way function mechanism works intreating liver fibrosis.
Methods: Male S-D rats were randomly divided into three groups: the control group(n=6) was injected with olive oil into abdominal cavity, cirrhotic animal model group(n=10) was treated in the same way with CCl_4(CCl_4/olive oil:v/v=1/1) 1ml/kg.Animals in Fuzheng Huayu group received compound intragastric administration inaddition of CCl_4 abdominal cavity injection. Eight weeks later, collagen was stainedby Masson-trichrome-staining and analyzed by image analysis software. Collagenfiber was half quantitively analyzed with image analysis software. After twodimensional gel electrophoresis, silver staining and analyzing gel image by softwarePDQUST 7.3, the diversity of protein points was analyzed with mass spectrometricanalysis.
Results: The degree of fibrosis of Fuzheng Huayu group was much slighter than thatof model group. The collagen deposited in liver decreased (8.9%±3.7% vs. 12.4%±4.7%, P＜0.05). Wiping off most albumin and immunoglobulin from plasma can getmore satisfying repeatability. In this research, we got 10 evidently diversityexpression proteins. In those proteins, plasma glutathione peroxidase and itsprecursor, prealbumin, haptoglobin, precusor of ApoA-Ⅳ, complement component4, inter-alpha-inhibitor H4 heavy chain and serine/threonine-protein kinase MARK1exhibited less expression in cirrhosis group and more expression in Fuzheng Huayugroup. The expression of proteins related to regeneration of liver and vimentinincreased in cirrhosis group and decreased in Fuzheng Huayu group.
Conclusions: Animal trial confirmed that Fuzheng Huayu Decoction had the functionof anti-fibrosis and anti-cirrhosis. The plasma proteomic study on the pharmaceuticalintervention of Fuzheng Huayu decoction explained the mechanism of its anti-fibrosisand anti-cirrhosis function. It is discovered that Fuzheng Huayu decoction mayfunction as anti-fibrosis agent through accelerating the protein composing,anti-oxidation, adjusting the cell proliferation and transformation. Proteomicsexplores a new way in the research of inspecting the curative effect and mechanism ofChinese traditional medicine compound.
研究背景与目的：肝硬化是临床常见的慢性进行性肝病，是我国疾病防治的主要任务之一。肝纤维化是肝硬化发展的必经病理过程。蛋白质组学应用各种技术手段从整体角度分析蛋白质组成、表达水平与修饰状态的动态变化，了解蛋白质之间的相互作用与联系，揭示蛋白质功能与细胞生命活动规律。疾病蛋白质组学为探讨疾病的发病机制、预防、早期诊断和治疗提供重要理论基础。本实验构建CCl_4诱导大鼠肝硬化模型，建立肝组织蛋白质组研究的技术平台；掌握进行大鼠肝组织双向凝胶电泳所需要的各种条件；了解CCl_4诱导大鼠肝硬化模型肝组织蛋白质组变化，为进一步研究肝纤维化、肝硬化发病机制奠定基础。
方法：将雄性S-D大鼠随机分成3组：正常对照组(n=6)给予橄榄油腹腔注射；肝硬化模型组(n=10)用CCl_4(CCl_4／橄榄油：v／v=1／1)按1ml／kg腹腔注射，每周2次，共8周诱导肝硬化模型。病理组织学观察肝纤维化严重程度并评分，用Masson三色染色法进行胶原染色。计数肝窦内α-SMA阳性细胞数观察肝星状细胞的活化情况。对肝组织进行双向凝胶电泳，应用PDQUST图像分析软件将肝硬化模型组与对照组肝组织2-DE胶图进行匹配，不同胶匹配点相对含量差异2倍及其以上的点为差异蛋白质点。对差异蛋白质点进行胶内消化、MALDI-TOF-TOF-MS质谱仪鉴定和数据库搜索确定差异蛋白质。用免疫印迹法验证双向凝胶电泳质谱技术鉴定的蛋白质表达改变。
结果：肝硬化模型组肝组织病理均提示典型纤维间隔和假小叶形成，α-SMA阳性细胞表达明显增加。肝组织双向电泳凝胶重复性满意，显示的蛋白斑点在分布、背景等方面基本一致。通过PDQUST软件分析的差异蛋白质点经胶内消化、质谱鉴定、蛋白质数据库搜索有33个点得到鉴定，分别属于28种蛋白质。在这些差异蛋白质中，有5种蛋白质仅在肝硬化模型组中出现；2种蛋白质仅在正常组出现；12种蛋白质在肝硬化模型组上调，9种蛋白质下调。这些蛋白质按其功能特性可分为与细胞增殖、能量代谢相关酶类、物质代谢和转运相关蛋白、细胞骨架和运动相关蛋白、氧化应激、内质网应激和热休克相关蛋白质。免疫印迹法进一步证实了蛋白质组技术显示的alpha crystallin B chain变化趋势。
结论：成功构建肝硬化大鼠动物模型。利用蛋白质组学技术，从整体角度将蛋白质变化综合一起，得到CCl_4诱导肝硬化大鼠肝组织蛋白质组的特征性变化。在CCl_4诱导大鼠肝硬化形成过程中，肝组织中差异表达的蛋白质可能通过影响细胞增殖、能量代谢、物质转运代谢、细胞骨架形成和运动、应激反应等参与、反映肝硬化的发生发展过程。
第二部分肝硬化模型血浆比较蛋白质组学研究
研究背景与目的：许多疾病可导致血浆中多种蛋白质改变，这种特征性变化对疾病诊断和疗效监测具有重要意义。迄今为止人类仅对少数血浆蛋白作为临床常规监测用于某些疾病的诊断。血浆蛋白质组的发展拓宽了对血浆蛋白质的认识。全面而系统地认识肝纤维化和肝硬化时血浆蛋白改变情况，将有助于肝纤维化、肝硬化的发病机制、诊断和药效研究。本实验将初步建立血浆蛋白质组技术平台；掌握去除大鼠血浆白蛋白和球蛋白的方法以及进行血浆双向凝胶电泳所需要的各种条件；了解CCl_4诱导大鼠肝硬化疾病模型的血浆蛋白质组变化，为进一步研究肝硬化的发病机制和诊断奠定基础。
方法：用CCl_4制备肝硬化动物模型，单纯橄榄油腹腔注射组作为对照组。造模8周后抽取血液，同组间血浆混匀，去除血浆中白蛋白和球蛋白。进行双向凝胶电泳，应用PDQUST图像分析软件对各种条件下除血浆白蛋白和球蛋白的效果进行评价。将肝硬化模型组与对照组血浆2-DE胶图进行匹配，不同胶匹配点相对含量的差异2倍及其以上的点为差异蛋白质点。对差异蛋白质点进行胶内消化、MALDI-TOF-TOF-MS质谱仪鉴定和数据库搜索鉴定差异蛋白质。用免疫印迹法验证双向凝胶电泳质谱技术所鉴定的蛋白质表达改变。
结果：使用Albumin／IgG Removal Kit可去除血浆中大部分白蛋白和球蛋白。去除后，双向凝胶电泳显示的蛋白质斑点由处理前的260±15个增加到598±42个。胶重复性满意，显示蛋白斑点在分布、背景等方面基本一致。通过PDQUST软件分析的差异蛋白质点经胶内消化、质谱鉴定、蛋白质数据库搜索有20个点得到鉴定，分别属于16种蛋白质：血浆谷胱甘肽过氧化物酶、谷胱甘肽过氧化物酶前体、载脂蛋白A-Ⅳ前体、载脂蛋白E、结合珠蛋白、前白蛋白、血浆视黄醇结合蛋白、α1-抗胰蛋白酶、微管相关调节激酶1、α1-巨球蛋白、补体4、α抑制剂H4重链、转铁蛋白、肝脏再生相关蛋白、波形纤维蛋白和RIKEN cDNA1700025816。免疫印迹法进一步证实了蛋白质组研究显示的结合珠蛋白变化趋势。
结论：去除大鼠血浆中的白蛋白和球蛋白可明显增加双向凝胶电泳蛋白质点的显示，优化了血浆2-DE图谱。本研究建立的血浆蛋白质组研究平台可用于疾病机制的研究。CCl_4诱导大鼠肝硬化模型血浆中细胞防御和免疫相关蛋白质、物质转运蛋白质、细胞骨架和转化调控蛋白、急性时相反应蛋白等在表达量上不同程度地发生了变化，提示这些蛋白质表达改变可能与肝硬化的发病机制有关，反映了肝硬化时血浆蛋白质的整体改变。
第三部分扶正化瘀方干预肝硬化大鼠血浆比较蛋白质组研究
研究背景与目的：运用蛋白质组学方法，分析中药复方对CCl_4诱导肝纤维化、肝硬化大鼠血浆与正常血浆蛋白质组差异表达，以期筛选出与药物作用相关的差异蛋白质，为肝纤维化、肝硬化的药物治疗机制和疗效评价提供依据。目前扶正化瘀方在治疗肝炎后肝硬化及慢性乙型肝炎肝纤维化方面取得良好的临床效果，其抗肝纤维化作用机制是多方面的，具有复方中药的多成分、多环节、多层次、多靶点作用特点，蛋白质组学为从整体水平研究扶正化瘀方开辟了新途径。以CCl_4大鼠肝纤维化模型作为研究对象，探讨肝纤维化过程中血浆蛋白质组表达改变，揭示扶正化瘀方对肝纤维化多途径作用机制及其疗效评价。
方法：将雄性S-D大鼠随机分为三组，正常对照组(n=6)给予橄榄油腹腔注射；肝硬化模型组(n=10)给予CCl_4(CCl_4／橄榄油：v／v=1／1)按1ml／kg腹腔注射，每周2次，共8周诱导肝硬化模型；扶正化瘀方组(n=10)在CCl_4腹腔注射同时给予扶正化瘀方灌胃。8周后通过Masson三色染色法用图像分析仪自动分析肝脏胶原面积。应用双向凝胶电泳技术经银染显色和PDQUST7.3软件分析凝胶图像，对三组中表达均有差异的蛋白质点用基质辅助激光解吸电离飞行时间串连质谱进行鉴定。
结果：扶正化瘀方干预组较模型组肝纤维化程度明显减轻，胶原纤维在肝内沉积减少(8.9％±3.7％vs.12.4％±4.7％，P＜0.05)。实验去除血浆中的高丰度蛋白质可获得重复性较好的血浆蛋白质图谱。共鉴定出10个明显差异表达的蛋白质。在这些蛋白质中，血浆谷胱甘肽过氧化物酶及其前体、前白蛋白、结合珠蛋白、载脂蛋白A-Ⅳ前体、补体4、α抑制剂H4重链和微管相关调节激酶1在肝硬化大鼠血浆中表达减少，扶正化瘀方干预组表达增加，肝脏再生相关蛋白和波形纤维蛋白在肝硬化大鼠血浆中表达增加，扶正化瘀方干预组表达减少。
结论：动物实验证实扶正化瘀方具有抗纤维化、肝硬化的作用。本实验初步探索了药物干预下的血浆蛋白质组研究，从血浆蛋白质整体变化角度阐述了扶正化瘀方抗肝纤维化和肝硬化作用，研究发现扶正化瘀方可能通过促进蛋白质合成、抗氧化、调控细胞增殖和转化等多方面发挥抗纤维化的作用。蛋白质组研究可能为复方中药疗效监测和作用机制研究开辟新途径。
Part one
Hepatic comparative proteomic study of cirrhotic rats induced by CCl_4Background and aims: Cirrhosis is a popular chronic progressive disease in China,and how to cure it is a major task of the Chinese medical specialists. Liver fibrosis isan indispensable pathological status during the development of cirrhosis. Proteomicsis a novel method to determine the constitution of proteins and to analyze the dynamicchanges of the expression and modification of proteins from an integrative level, bywhich the interaction and relationships among proteins are studied and the functionsof proteins and the principles of cellular activities are discovered. Disease proteomicsaims to lay theoretic foundation for studying the mechanisms, prevention,pre-diagnosis and therapy of diseases. In the present study a rat model ofCCl_4-induced cirrhosis was constructed, a platform for proteome of liver tissue wasbuilt up, and the conditions for two-dimensional gel electrophoresis of rat liver tissuewere determined. By analyzing the proteome of the rat model of CCl_4-inducedcirrhosis the study laid a basis for the further research of liver fibrosis, and cirrhosis.Methods: Male S-D rats were randomly divided into three groups: the control group(n=6) was injected with olive oil into abdominal cavity, cirrhotic animal model group(n=10) was treated in the same way with CCl_4(CCl_4/olive oil:v/v=1/1) 1ml/kg twicea week and for 8 weeks. The degree of liver fibrosis and inflammation grade wasobserved using histopathology study, collagen were visualized by Masson-trichrome--staining.α-SMA positive cells in hepatic sinus were counted to observe the activationof hepatic stellate cell. After two-dimensional gel electrophoresis the gels fromcirrhosis group and control group were analyzed by image analysis softwarePDQUST to match same points. Twice or more different content were considereddifferent points. Those points were digested, analyzed with MALDI-TOF-TOF-MSmass spectrograph and searched in protein database. The expression of protein wasidentified by immunoblotting test.
Results: Typical fibrotic septa and pseudo-lobule were observed in cirrhotic animal model. The expression ofα-SMA positive cell increased. The reproducibility wassatisfying, proteins had approximately the same distribution and background. Afterprotein digestion in situ and mass spectrometric analysis, 33 points corresponding to28 proteins were identified. In those different proteins, 5 proteins only appear in livercirrhotic animal model group, 2 proteins only appear in control group. 12 proteinsexpression was promoted and 9 depressed in cirrhotic animal model group. Thoseproteins function in cell proliferation, energy metabolization, substancemetabolizetion and transport of acting enzyme, cytoskeleton and protein movement,oxidation stress, endoplasmic reticulum stress and hot shock stress respectively. Thechange trend of alpha crystalline B chain was further approved by immunoblottingtest.
Conclusions: We successfully established a cirrhotic rat model. The change of thoseproteins was considered integratively, from which we obtained the characteristicchange in the liver tissue of cirrhotic animal induced by CCl_4. On the progress ofcirrhosis, the expression of diverse proteins may participate in the occurring anddeveloping of cirrhosis in the ways of cell proliferation, energy metabolization,substance metabolization, cytoskeleton structure and protein movement, and stressrespond.
Part two
Plasma comparative proteomic study of cirrhotic rats induced by CCl_4Background and aims: Many diseases may affect proteins in plasma. Thecharacteristic changes are important to diagnosing diseases and monitoring curativeeffect. So far, only few plasma proteins are used in clinic as markers of some diseases.With the development of plasma proteome, more plasma proteins are entering ourview. Studying the change of plasma proteins of hepatic fibrosis and cirrhosis helps tounderstand the pathologic process, diagnoses and curative effect. In this research, weestablished a plasma proteomic technologic platform, on which we tried to establishthe methods of eliminating rat plasma albumin and globulin, and to optimize theconditions forplasma two-dimensional gel electrophoresis. It can be served as a basisfor further researches in cirrhosis by studying the change of plasma proteome of CCl_4-induced rat cirrhostic model.
Methods: Cirrhotic rats were induced by CCl_4 injection to abdominal cavity, whileolive oil was administrated in the control group. Eight weeks after injection, bloodwas collected and plasma from the same group was mixed. Albumin andimmunoglobulin were wiped off. After two-dimensional gel electrophoresis, gel mapswere analyzed with PDQUST. Points from model group and control group werematched, and twice or more different content were considered different points. Thosepoints were digested, analyzed with MALDI-TOF-TOF-MS mass spectrograph andsearched from protein database. The expression of protein was identified byimmunoblotting test.
Results: Albumin/IgG Removal Kit can wipe off most of albumin andimmunoglobulin from plasma. After the wiping, the number of gel map protein pointsincreases from 260±15 to 598±42. The repeatability was satisfying, and proteins hadapproximately same distribution and background. After protein digestion in situ andmass spectrometric analysis, 20 points are identified which belongs to 16 kinds ofproteins: plasma glutathione peroxidase, glutathione peroxidase precursor,apolipoprotein A-Ⅳprecursor, apolipoprotein E, haptoglobin, prealbumin,retinol-binding protein, alpha-1-antitrypsin, serine/threonine-protein kinase MARK1,alpha-1-Macroglobulin, complement component 4, inter-alpha inhibitor H4 heavychain, transferring, liver regeneration-related protein LRRG03, vimentin, RIKENcDNA1700025B16. The change trend of haptoglobin was further approved byimmunoblotting test.
Conclusions: Wiping off albumin and immunoglobulin from plasma can increase theprotein points display obviously, and optimize the plasma 2-DE map. The platformestablished in our study can be used in studying disease mechanism. In the liver ofcirrhotic rat model induced by CCl_4, those protein participate in cell defense andimmunity, protein transport and cytoskeleton and transformation, and acute phasereactive proteins. This hint the change of protein may be induced by the pathogenesis ofcirrhosis, those protein may take part in diverse ways during the occurring anddeveloping of cirrhosis.
Part three
The effect of Fuzheng Huayu Decoction on plasma proteome of cirrhotic rats Background and aims: Using proteomics we studied the role of Chinese traditionalmedicine compound on the differential expression of proteins in liver fibrosis andcirrhosis induced by CCl_4. The aim is to find the proteins involved in the certainphysiological and pathological progress, and to lay foundation for the early diagnoseand therapy for liver fibrosis and cirrhosis. Presently, Chinese traditional medicine hasshown obviously advantage in treatment of liver fibrosis. Fuzheng Huayu Decoctionhas nice clinic result in treating post-hepatitis cirrhosis and chronic hepatitis B liverfibrosis. The antifibrotic mechanism is various, and the compound works in amulti-component, multi-step, multi-arrangement, multi-target way. Proteomics opensup a new way in researching Fuzheng Huayu Decoction from an integrative level. Westudied the change of plasma proteome in the progress of cirrhosis in rat modelinduced by CCl_4, and tried to find how the multi-way function mechanism works intreating liver fibrosis.
Methods: Male S-D rats were randomly divided into three groups: the control group(n=6) was injected with olive oil into abdominal cavity, cirrhotic animal model group(n=10) was treated in the same way with CCl_4(CCl_4/olive oil:v/v=1/1) 1ml/kg.Animals in Fuzheng Huayu group received compound intragastric administration inaddition of CCl_4 abdominal cavity injection. Eight weeks later, collagen was stainedby Masson-trichrome-staining and analyzed by image analysis software. Collagenfiber was half quantitively analyzed with image analysis software. After twodimensional gel electrophoresis, silver staining and analyzing gel image by softwarePDQUST 7.3, the diversity of protein points was analyzed with mass spectrometricanalysis.
Results: The degree of fibrosis of Fuzheng Huayu group was much slighter than thatof model group. The collagen deposited in liver decreased (8.9%±3.7% vs. 12.4%±4.7%, P＜0.05). Wiping off most albumin and immunoglobulin from plasma can getmore satisfying repeatability. In this research, we got 10 evidently diversityexpression proteins. In those proteins, plasma glutathione peroxidase and itsprecursor, prealbumin, haptoglobin, precusor of ApoA-Ⅳ, complement component4, inter-alpha-inhibitor H4 heavy chain and serine/threonine-protein kinase MARK1exhibited less expression in cirrhosis group and more expression in Fuzheng Huayugroup. The expression of proteins related to regeneration of liver and vimentinincreased in cirrhosis group and decreased in Fuzheng Huayu group.
Conclusions: Animal trial confirmed that Fuzheng Huayu Decoction had the functionof anti-fibrosis and anti-cirrhosis. The plasma proteomic study on the pharmaceuticalintervention of Fuzheng Huayu decoction explained the mechanism of its anti-fibrosisand anti-cirrhosis function. It is discovered that Fuzheng Huayu decoction mayfunction as anti-fibrosis agent through accelerating the protein composing,anti-oxidation, adjusting the cell proliferation and transformation. Proteomicsexplores a new way in the research of inspecting the curative effect and mechanism ofChinese traditional medicine compound.
引文
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