权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Inactivation of Hepatic Stellate Cells During Reversal of Liver Fibrosis

肝纤维化逆转过程中肝星状细胞失活

基本信息

批准号：
9271180
负责人：
Tatiana Kisseleva
金额：
$ 33.71万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-15 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9271180
关键词：
Acetylation Adult Affect Binding Sites Biochemical Biology Cell model Cells ChIP-seq Chronic Cirrhosis Collagen Collagen Type I DNA Binding Data Deposition Detection Development Epigenetic Process Event Extracellular Matrix Proteins Flow Cytometry Gene Activation Gene Chips Gene Expression Gene Expression Profile Gene Targeting Genes Genetic Transcription Goals Healthcare Hepatic Stellate Cell Hepatotoxicity Human Human Genome In Vitro Injury to Liver Investigation Knockout Mice Label Link Liver Liver Fibrosis Liver diseases Location LoxP-flanked allele Maintenance Mediating Messenger RNA Molecular Mus Myofibroblast PPAR gamma Pathway interactions Patients Phenotype Population Production Recovery Regulation Regulator Genes Repression Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction Site Small Interfering RNA Source Stimulus System TIMP1 gene Tamoxifen Testing Time Translating Transplantation Up-Regulation base blocking factor care burden genome wide methylation genome-wide histone modification in vitro testing in vivo injured insight interest intrahepatic knock-down liver injury mRNA Expression mouse genome mouse model novel overexpression prevent public health relevance pup response therapeutic target transcription factor transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): Chronic liver injury results in hepatic fibrosis, in which quiescent hepatic stellate cells (qHSCs) activate into myofibroblasts, depositing extensive extracellular matrix (ECM) proteins. Cessation of fibrogenic stimuli often results in regression of liver fibrosis and is associated with the disappearance of activated HSCs (aHSCs)/myofibroblasts. We have recently demonstrated that some aHSCs apoptose, while other aHSCs inactivate (iHSCs) into a quiescent-like phenotype. The overall goals of this Project are to identify the molecular factors that may prevent HSC activation into myofibroblasts, or that revert aHSC into an inactivated state. Our central hypothesis is that genome wide epigenetic changes regulate HSC phenotype by activation (or suppression) of transcriptional activity in HSCs. We also hypothesize that activation of PPARγ-target genes regulates quiescent and inactivated HSC phenotypes. AIM1: We will assess the genome wide methylation and acetylation sites using ChIP-Seq in qHSCs, aHSCs and iHSCs in order to identify motifs and transcription factors critical for HSC inactivation. Using in vitro systems, we will determine f siRNA knock-down or overexpression of these factors blocks HSC activation, or triggers HSC inactivation. AIM2: We have demonstrated that PPARγ is re-expressed in HSCs during inactivation. To gain a greater insight into the mechanisms of HSC inactivation, we will conduct a broad investigation of the epigenetic changes that regulate PPARγ-target genes in distinct HSC phenotypes (qHSCs, aHSCs and iHSCs). We will test if functional inhibition of these genes affects the qHSC and iHSC phenotypes. AIM3: We will assess the role of PPARγ and PPARγ target genes in HSC biology in vivo using knockout mice, in which PPARγ is constitutively or inducibly deleted specifically in HSCs. A specific role of PPARγ in the maintenance of quiescent HSC phenotype and inactivation of aHSCs will be assessed in PPARγ-deficient HSCs versus wild type HSCs. We anticipate that the collective results obtained in AIMs 1-3 will identify specific factors that can revert aHSCs into an inactivated quiescent-like state. AIM4: Our findings in mice must be translated to patients with fibrotic liver disease. To examine how human HSCs inactivate, quiescent human HSCs will be engrafted into livers of Rag2-/-γc-/- mice, and their activation will be induced in vivo by CCl4 followed by recovery. Human qHSCs, aHSCs and iHSCs will be isolated at each time point and analyzed by RT-PCR, Human genome microarray and flow cytometry. Next, human iHSCs will be subjected to ChIP-Seq analysis, and inactivation-specific targets will be identified (and compared to that in mouse iHSCs). The ability of these targets to inactivate human aHSCs into a quiescent-like state will be tested in vitro and in vivo using siRNA knock-down or intrahepatic transplantation of human HSCs into Rag2-/-γc-/- mice. The results of our studies will give new insight into mechanisms underlying HSC inactivation in mice and patients, identifying potential therapeutic targets that can induce inactivation of activated HSCs/myofibroblasts in fibrotic liver.

描述（由申请人提供）：慢性肝损伤导致肝纤维化，其中静止的肝星状细胞（qHSC）活化为肌成纤维细胞，沉积大量细胞外基质（ECM）蛋白。纤维化刺激的停止通常导致纤维化的消退。肝纤维化和与活化的HSC（aHSC）/肌成纤维细胞的消失有关。我们最近已经证明，一些aHSC凋亡，而其他aHSC凋亡（iHSC）成静止样表型。本项目的总体目标是确定可能阻止HSC活化成肌成纤维细胞或使aHSC恢复至失活状态的分子因子。我们的中心假设是，基因组范围的表观遗传变化调控HSC表型激活（或抑制）的转录活性的HSC。我们还假设，激活的PPARγ靶基因调节静止和失活的HSC表型。目标1：我们将使用ChIP-Seq评估qHSC、aHSC和iHSC中的全基因组甲基化和乙酰化位点，以鉴定对HSC失活至关重要的基序和转录因子。使用体外系统，我们将确定这些因子的siRNA敲低或过表达阻断HSC活化或触发HSC失活。AIM 2：我们已经证明，在灭活过程中，HSC中的PPARγ重新表达。为了更深入地了解HSC失活的机制，我们将对不同HSC表型（qHSC、aHSC和iHSC）中调节PPARγ靶基因的表观遗传变化进行广泛研究。我们将测试这些基因的功能抑制是否影响qHSC和iHSC表型。目标3：我们将使用基因敲除小鼠评估PPARγ和PPARγ靶基因在体内HSC生物学中的作用，其中PPARγ在HSC中特异性地组成性或诱导性缺失。将在PPARγ缺陷型HSC与野生型HSC中评估PPARγ在维持静止HSC表型和aHSC失活中的特定作用。我们预计，在AIM 1-3中获得的集体结果将确定可以使aHSC恢复到失活的静止样状态的特定因素。目标4：我们的在小鼠中的发现必须转化为患有纤维化肝病的患者。为了检查人HSC如何活化，将静止的人HSC移植到Rag 2-/-γc-/-小鼠的肝脏中，并通过CCl 4在体内诱导其活化，然后恢复。将在每个时间点分离人qHSC、aHSC和iHSC，并通过RT-PCR、人基因组微阵列和流式细胞术进行分析。接下来，将对人iHSC进行ChIP-Seq分析，并鉴定失活特异性靶点（并与小鼠iHSC中的靶点进行比较）。的能力将使用siRNA敲低或将人HSC肝内移植到Rag 2-/-γc-/-小鼠中，在体外和体内测试这些靶点中将人aHSC转化为静止样状态的能力。我们的研究结果将为小鼠和患者HSC失活的潜在机制提供新的见解，确定潜在的治疗靶点，可以诱导纤维化肝脏中活化的HSC/肌成纤维细胞失活。