Determining how chronic ETOH influences the regenerative activities of hepatocyte subpopulations

确定慢性 ETOH 如何影响肝细胞亚群的再生活动

• 批准号: 10297361
• 负责人: Hao Zhu
• 金额: $ 54.81万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10297361
• 关键词: Alcoholic Liver Diseases; Alcohols; CRISPR screen; Cell Proliferation; Cell physiology; Cells; Chronic; Contracts; Data; Disease; Enzymes; Ethanol; Exhibits; Exposure to; FRAP1 gene; Frequencies; Genes; Genetic Transcription; Hepatic; Hepatocyte; Homeostasis; Human; Impairment; Injury; Iron; Iron Overload; Iron Uptake Inhibition; Label; Liver; Liver Regeneration; Lobular; Metabolic; Modeling; Morbidity - disease rate; Natural regeneration; Pathogenesis; Patients; Population; Production; Proliferating; Reactive Oxygen Species; Regulation; Source; Time; Tissues; gain of function; healing; hepcidin; in vivo; liver cell proliferation; liver injury; mortality; non-alcoholic fatty liver disease; overexpression; regenerative; response; single-cell RNA sequencing; success; tissue repair

PROJECT SUMMARY Alcoholic liver disease (ALD) is exacerbated by impaired liver regeneration. The cellular basis of liver regeneration is unclear and whether hepatocytes in different zones differ in regenerative activity is unclear, in part because fate-mapping has only been performed on a few hepatocyte subsets. The liver is organized into zones in which hepatocytes express different metabolic enzymes. To systematically compare the regenerative activities of these distinct subsets of hepatocytes, we developed twelve new CreER strains. Lineage tracing during normal homeostasis showed that cells from periportal zone 1 and pericentral zone 3 contracted in number, while cells from mid-lobular zone 2 expanded in number. Hepatocytes in different regions of the liver thus exhibit differences in turnover and zone 2 is an important source of new hepatocytes during homeostasis. Because zone 2 may represent a reserve population sheltered from pericentral and periportal liver injuries, we hypothesize that these cells also preferentially repopulate livers exposed to modest chronic injuries such as alcohol. Our preliminary scRNA-seq and in vivo CRISPR screens identified two critical zone 2 specific genes that regulate zone 2 hepatocyte proliferation and survival: Igfbp2 and Hamp2. Both of these secreted factors are suppressed in NAFLD and ALD in humans, which suggests functional importance in disease. Igfbp2 operates through mTOR and Ccnd1 to promote zone 2 hepatocyte proliferation. Hamp1 and Hamp2 encode for hepcidins, which negatively regulate iron uptake by inhibition of iron transporters and thus protects the body from iron overload. Patients with ALD accumulate hepatic iron through suppression of hepcidins. Free iron enhances reactive oxygen species (ROS) production in the liver, leading to alcohol-induced liver injury. We hypothesize that ALD pathogenesis is accelerated through the suppression of Igfbp2 and Hamp1/2, and that this involves changes in the number or function of zone 2 hepatocytes. In Aim 1, we will first use our CreER models to systematically determine the extent to which zone 2 cells repopulate the liver in the setting of ETOH. To understand how zone 2 cell might be involved in regeneration after ETOH, we will perturb two critical zone 2 genes using loss and gain of function approaches. In Aim 2, we will ask if Igfbp2 is necessary and sufficient to regulate the frequency or repopulating activities of zone 2 cells in the context of ETOH. In Aim 3, we will ask if Hamp1 and Hamp2 are necessary and sufficient to regulate the frequency or repopulating activities of zone 2 cells. Success in this project will for the first time define the cellular basis of regeneration in response to ETOH, allow us to focus on critical subpopulations, and determine the importance of two critical zone 2 specific genes in ALD.

项目摘要 酒精性肝病（ALD）因肝脏再生受损而加剧。肝脏的细胞基础 再生尚不清楚，不同区域中的肝细胞在再生活性上是否有所不同，尚不清楚 部分是因为仅在几个肝细胞子集上进行命运映射。肝脏被组织成 肝细胞表达不同代谢酶的区域。系统地比较再生 这些独特的肝细胞子集的活动，我们开发了十二种新的Creer菌株。谱系跟踪 在正常的体内平衡期间，表明外围区1和周围区域3的细胞数量收缩， 而来自叶2区的细胞数量扩大。肝脏不同区域的肝细胞因此表现出 营业额和2区的差异是稳态期间新肝细胞的重要来源。因为 第2区可能代表避免腹膜和周围肝损伤的储备人群，我们假设 这些细胞还优先将暴露于适度的慢性损伤（例如酒精）的肝脏重生。我们的 初步的SCRNA-SEQ和INTECR CRISPR屏幕鉴定了两个调节的两个临界区2特定基因 区2肝细胞增殖和生存：IGFBP2和HAMP2。这两个分泌的因素都被抑制 在人类的NAFLD和ALD中，这表明在疾病中具有功能重要性。 IGFBP2通过MTOR运行 和CCND1促进2区2肝细胞增殖。 hamp1和hamp2编码肝素，其中 通过抑制铁转运蛋白对铁的摄取进行负面调节，从而保护人体免受铁超载。 ALD患者通过抑制肝素积聚肝铁。游离铁增强反应性 肝脏中的氧（ROS）产生，导致酒精诱导的肝损伤。我们假设那个ALD 通过抑制IGFBP2和HAMP1/2加速发病机理，这涉及变化 区域2肝细胞的数量或功能。在AIM 1中，我们将首先使用我们的Creer模型系统地 确定2区2细胞在ETOH环境中重新填充肝脏的程度。了解区域如何 2细胞可能参与ETOH后的再生，我们将使用损失和增益扰动两个临界区2基因 功能方法。在AIM 2中，我们将询问IGFBP2是否需要和足以调节频率或 在ETOH的背景下，区域2细胞的重新流动活性。在AIM 3中，我们将询问Hamp1和Hamp2是否是 必要且足够调节2区细胞的频率或重新流动活性。成功 项目将首次定义对ETOH的再生细胞基础，让我们专注于 关键亚群，并确定ALD中两个关键区2特定基因的重要性。