Identification of Serum Markers of Liver Fibrogenesis/ Fibrolysis by Proteomics

描述（由申请人提供）：肝纤维化通常进展为肝硬化。肝硬化的演变是缓慢的（几十年），通过频繁的肝活检监测进展既不道德，也容易发生采样错误。纤维化是由于纤维溶解（基质的去除）的动态过程的失衡而有利于纤维发生（基质的沉积）。目前还没有非侵入性的指标来衡量肝纤维化和纤维溶解。由于缺乏此类标志物，因此无法量化肝脏患者进展为肝硬化的个体风险，或开发可抑制进展或诱导纤维化逆转的经证实的抗纤维化药物。 在我们的前期工作中，我们建立了继发性胆汁性肝硬化和硫代乙酰胺中毒引起的全小叶性肝硬化大鼠的进展模型。这些动物的肝硬化分别在胆消化吻合术和抗纤维化剂常山酮后逆转。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，我们发现了第一个与纤维化和纤维溶解相关的血清蛋白。 我们假设，通过使用具有肝纤维化进展或逆转的同质大鼠组，我们可以1。将差异血清蛋白质组学模式与如在配对肝脏样品中验证的肝纤维化发生或纤维溶解的活性相关联，以及2.鉴定差异表达蛋白。为了实现这些目标，我们追求以下目标：1。彻底表征我们的大鼠模型的胆道和全小叶纤维化进展和逆转的动力学，2.应用定量蛋白质组学和同量异序蛋白质标签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）种不同的同量异序肽标记物（iTRAQ）连接到来自代表去除大量血清蛋白后肝纤维化和纤维溶解演变的组的4个分级血清池的胰蛋白酶上。将通过2D纳米液相色谱和MALDI-TOF/TOF质谱鉴定差异表达的蛋白质。基于这一建议的发现，将在未来的应用中开发用于门脉与小叶纤维化和纤维溶解的血清标志物的ELISA。对人类蛋白质组的适应和前瞻性验证将允许无创监测肝病患者的纤维化进展和消退。

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.