Identification of Serum Markers of Liver Fibrogenesis/ Fibrolysis by Proteomics

通过蛋白质组学鉴定肝纤维发生/纤维溶解的血清标志物

• 批准号: 7313389
• 负责人: DETLEF SCHUPPAN
• 金额: $ 21.25万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-06 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7313389
• 关键词: Accounting; Alcoholic Liver Cirrhosis; Anastomosis - action; Animals; Biliary; Biliary cirrhosis; Cessation of life; Characteristics; Cirrhosis; Collagen; Collection; Data; Deposition; Digestion; Disease regression; Enzyme-Linked Immunosorbent Assay; Evolution; Excision; Fibrosis; Future; Gene Expression; Generations; Goals; Halofuginone; Hepatic; Hepatic Fibrogenesis; Hospitals; Human; Individual; Intoxication; Label; Ligation; Liquid Chromatography; Liver; Liver Fibrosis; Liver diseases; Lobular; Mass Spectrum Analysis; Measures; Methodology; Modeling; Molecular Profiling; Monitor; Obstructive Liver Cirrhosis; Patients; Pattern; Peptides; Pharmaceutical Preparations; Prevalence; Process; Proteins; Proteome; Proteomics; Rattus; Recovery; Risk; Running; Sampling; Sampling Errors; Serum; Serum Markers; Serum Proteins; Staging; Standards of Weights and Measures; Testing; Thioacetamide; Time; Transcript; Trypsin; Validation; Week; Work; Wound Healing; base; bile duct; drug development; fibrogenesis; liver biopsy; nano; prospective; response; size; time interval

DESCRIPTION (provided by applicant): Liver fibrosis often progresses to cirrhosis. The evolution of cirrhosis is slow (decades) and monitoring of progression by frequent liver biopsies is both unethical and subject to sampling error. Fibrosis results from a dysbalance of the dynamic processes of fibrolysis (removal of matrix) in favor of fibrogenesis (deposition of matrix). There exist no noninvasive markers to measure hepatic fibrogenesis and fibrolysis. Due to the lack of such markers it has been impossible to quantify the individual risk of liver patients to progress to cirrhosis, or to develop proven antifibrotic drugs that can inhibit progression or induce fibrosis reversal. In our preliminary work we established models of progression in rats with secondary biliary cirrhosis and with panlobular cirrhosis due to thioacetamide intoxication. Cirrhosis in these animals reverses after biliodigestive anastomosis and the antifibrotic agent halofuginone, respectively. We defined specific liver gene expression profiles associated with fibrosis progression and reversal. By applying advanced serum proteomics we found first serum proteins associated with fibrogenesis and fibrolysis. We hypothesize that by using homogeneous groups of rats with progression or reversal of liver fibrosis, we can 1. relate differential serum proteomic patterns to the activity of hepatic fibrogenesis or fibrolysis as verified in the paired liver samples, and 2. identify the differentially expressed proteins. To reach these goals we pursue the following aims: 1. to thoroughly characterize the dynamics of our rat models of biliary and panlobular fibrosis progression and reversal, 2. to use quantitative proteomics with isobaric protein tags to identify serum markers of hepatic fibrogenesis and fibrolysis. To achieve these aims, we will attach 4 (8) different isobaric peptide labels (iTRAQ) to trypsin digests of 4 pools of fractionated sera from groups representing the evolution of hepatic fibrogenesis and fibrolysis after removal of abundant serum proteins. Differentially expressed proteins will be identified by 2D Nano-liquid chromatography and MALDI-TOF/TOF mass spectrometry. Based on the findings of this proposal, ELISAs for serum markers of portal vs. lobular fibrogenesis and fibrolysis will be developed in a future application. Adaptation to the human proteome and prospective validation shall allow noninvasive monitoring of fibrosis progression and regression in patients with liver diseases.

描述(申请人提供)：肝纤维化通常进展为肝硬变。肝硬变的发展是缓慢的(几十年)，通过频繁的肝活检来监测进展是不道德的，而且容易受到抽样错误的影响。纤维化是纤维溶解(基质去除)有利于纤维化形成(基质沉积)的动态过程的失衡所致。目前尚无非侵入性的标记物来衡量肝纤维化的形成和纤溶。由于缺乏这些标记物，一直不可能量化肝脏患者进展为肝硬变的个体风险，也不可能开发出经过验证的能够抑制进展或诱导纤维化逆转的抗纤维化药物。 在我们的前期工作中，我们建立了继发性胆汁性肝硬变和硫代乙酰胺中毒所致的泛小叶性肝硬变大鼠的进展模型。这些动物的肝硬变分别在胆管胃肠道吻合和抗纤维化药物常压呋喃酮治疗后逆转。我们定义了与纤维化进展和逆转相关的特定肝脏基因表达谱。通过应用先进的血清蛋白质组学，我们首次发现了与纤维形成和纤维溶解相关的血清蛋白质。 我们假设，通过使用肝纤维化进展或逆转的同质组大鼠，我们可以1.将不同的血清蛋白质组模式与配对肝脏样本中证实的肝纤维化或纤维溶解的活性联系起来，2.鉴定差异表达的蛋白质。为了达到这些目标，我们追求以下目标：1.彻底描述我们的大鼠胆管和泛小叶纤维化进展和逆转模型的动力学特征，2.使用等压蛋白质标记的定量蛋白质组学来鉴定肝纤维化和纤维溶解的血清标志物。 为了实现这些目标，我们将4(8)个不同的等压肽标记(ITRAQ)附加到4个分离血清的胰酶消化液中，这些分离血清来自代表肝纤维化和去除大量血清蛋白后的纤维溶解的演变。差异表达的蛋白质将通过2D纳米液色谱和MALDI-TOF/TOF质谱仪进行鉴定。基于这一建议的发现，门静脉与小叶纤维化和纤溶的血清标志物的ELISA将在未来的应用中得到开发。对人类蛋白质组的适应和前瞻性验证将允许对肝病患者的纤维化进展和消退进行非侵入性监测。