肝脏再生中转录因子动态变化的研究

The liver is an important organ in the body that has a central role in metabolic homeostasis, as it is responsible for the synthesis, metabolism, and storage. Meanwhile, the liver is the main detoxifying organ of the body and is likely to be injured by metabolic or exogenous toxins. The powerful regeneration capacity of the liver is a logical adaptation in evolution. The process of liver regeneration undergoes priming, proliferation and termination with the precise control in each step. The numerous cytokine and transcription factor mediated pathways that are involved in regulating liver regeneration. Even though some transcription factors such as AP1, STAT3 and NF B were reported to play essential roles in liver regeneration, as a crucial biological event that coordinates a variety of regulatory pathways, little is known about how this complex transcriptional network progresses in a multi-step manner up to date. .Transcription factors (TF) are families of proteins that bind to specific DNA sequences (Transcription Factors Response Elements, TFRE) and function as regulators of many cellular processes. Due to the low abundance of TFs, direct measurement of TFs on a proteome scale remains a challenge. To this end, we have developed a large-scale endogenous TFs enrichment and identification strategy with affinity reagent containing a concatenated tandem array of the consensus TFREs (catTFRE) of most transcription factor families. Using this catTFRE to enrich TFs from cells and tissues, and combining with protein/peptide differential fractionation for protein identification with mass spectrometry, we were able to identify 200 to 400 transcription factors from a single cell line or tissue in one experiment set, which accounts for most of the TFs that are expressed and can be detected on the mRNA level. Incorporating a quantitative mass spectrometry strategy, this method is able to measure proteome-wide TFs changes in different biological processes or under exogenous stimulation. .In this project, we will apply this powerful quantitative TF screening strategy on the in-depth description of dynamic TF alteration in liver regeneration induced by 2/3 liver hepatectomy. The change of TF pattern in liver regeneration will be monitored and comprehensively compared by combining our TFRE strategy with quantitative proteomics. A set of potential TF candidates will be further validated. Our study will uncover the dynamic change of TF on protein level in a large-scale proteome aspect, which eventually expends people's knowledge of transcription factor network in liver regeneration.

肝脏具有机体唯一的器官水平再生能力。肝脏再生参与了多种肝脏损伤的重建，如肝脏纤维化、肝脏手术切除、病毒性肝炎等，是肝脏维持形态和功能的主要保障。肝脏再生的过程涉及极为复杂的转录因子调控体系，但目前转录因子变化及机制还很不清楚。我们以串联多拷贝的转录因子结合序列（Transcription Factor Response Element, TFRE）为诱饵，在细胞中大规模的富集内源性转录因子，结合定量质谱技术，可在一种细胞中同时鉴定和定量多达几百种转录因子。本项目拟在此基础上开展深入研究 ，对在肝脏再生过程的各时间和事件节点中转录因子进行鉴定，对其动态变化进行定量，最终描绘出肝脏再生过程中各类转录因子在蛋白水平上的动态变化网络。研究将建立肝脏再生与转录因子谱之间的联系，发现一系列参与肝脏再生的重要转录因子，获得极具基础研究和应用价值的肝脏再生转录因子候选靶点。

肝脏是机体内最大的消化和代谢器官，承担着代谢、合成、营养的储存和重新分配。肝脏分泌大量数目的血清蛋白，包括白蛋白、急性期蛋白、酶类和共刺激分子等，是机体内最重要的解毒器官，具有很强的胆汁分泌和毒物分解代谢的功能。肝脏不仅承担着代谢和解毒的功能，同时还肩负清除肠道来源的外来物质的任务。肝脏在此过程中受到各种因素刺激，产生损伤，如酒精性肝炎，非酒精性肝炎，病毒性肝炎，化学和代谢毒物诱导的肝损伤，外伤和手术等。幸运的是，肝脏具有机体唯一的器官水平再生能力。肝脏再生参与了多种肝脏损伤的重建，如肝脏纤维化、肝脏手术切除、病毒性肝炎等，是肝脏维持形态和功能的主要保障。人和小鼠基因组中约6%的基因编码转录因子，是人类基因组编码的第二大类蛋白。转录因子不仅在基因表达调控中扮演重要的角色，而且是细胞内信号转导网络的关键节点，细胞感受胞内胞外刺激的各种信号通路通过转录因子相互交联，从而形成一个复杂的信号网络。肝脏再生的过程涉及极为复杂的转录因子调控体系，但目前转录因子变化及机制还很不清楚。. 在该课题中，我们以串联多拷贝的转录因子结合序列（Transcription Factor Response Element, TFRE）为诱饵，发明了内源性转录因子分析策略。该策略可在细胞中大规模的富集内源性转录因子，结合定量质谱技术，可在一种细胞中同时鉴定和定量多达几百种转录因子，达到RNA-seq的覆盖水平。为了进一步提升转录因子鉴定效率，我们还开发了双反向蛋白质组快速扫描平台Fast-seq，该平台可在12小时内完成8000-10000个基因产物的高通量鉴定和定量。TFRE和Fast-seq的综合应用可实现对各种组织器官细胞转录因子的全景式覆盖。最后，我们利用TFRE和Fast-seq联合技术对在肝脏再生过程的各时间和事件节点中转录因子进行鉴定，对其动态变化进行定量，共在小鼠肝脏再生各时期（启始、发展、终止）鉴定和定量出400多种转录因子，最终描绘出了肝脏再生过程中各类转录因子在蛋白水平上的动态变化网络。本课题建立了一系列的蛋白质组技术平台、转录因子大规模富集和鉴定技术，搭建了肝脏再生与转录因子谱之间的对应关系，发现一系列参与肝脏再生的重要转录因子，获得极具基础研究和应用价值的肝脏再生转录因子候选靶点。