Impact of hepatocyte lineage life history dynamics on liver homeostasis in the ag

肝细胞谱系生命史动态对肝脏稳态的影响

• 批准号: 8707923
• 负责人: Tomas Gedeon
• 金额: $ 31.11万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8707923
• 关键词: Acetaminophen; Adolescent; Adult; Affect; Age; Age-Months; Aging; Aging-Related Process; Albumins; Aneuploidy; Animal Model; Animals; Apoptotic; Belief; Birth; Birth Rate; Cell Aging; Cell Cycle; Cell Lineage; Cell Nucleus; Cell Proliferation; Cell Transplants; Cells; Cessation of life; Chronic; Chronic stress; Communities; Complex; Computer Simulation; DNA Adducts; Data; Deterioration; Development; Diet; Diploidy; Environment; Event; Exhibits; Fatty acid glycerol esters; Flow Cytometry; Fluorescence; Future; Gene Expression; Genetic Markers; Growth; Half-Life; Health; Hepatic; Hepatocyte; Homeostasis; Human; International; Intrinsic factor; Investigation; Length; Liver; Longevity; Maintenance; Mammals; Measures; Metabolic stress; Methods; Microsatellite Repeats; Mitosis; Modeling; Molecular; Mus; Mutation; Natural regeneration; Newborn Infant; Normal Cell; Obesity; Organ; Organism; Oxidative Stress; Partial Hepatectomy; Pharmaceutical Preparations; Phenobarbital; Physiological; Physiology; Play; Ploidies; Polyploidy; Population; Process; Process Measure; Production; Proliferating; Property; Protocols documentation; Recording of previous events; Research; Resistance; Resource Sharing; Rest; Role; Staging; Stem cells; Stress; Subgroup; System; Testing; Time; Tissues; Toxic effect; Toxin; age related; aged; animal data; base; cell age; cell type; design; embryonic stem cell; in vivo; innovation; life history; liver function; liver injury; mouse model; novel; oval cell; progenitor; reconstitution; regenerative; research study; response; self-renewal; simulation; stem; stem cell population; telomere; tool

DESCRIPTION (provided by applicant): We developed systems to investigate hepatocyte lineage life history dynamics in vivo. We propose to define the factors that determine hepatocyte lineage birth-rates and longevities, and to describe their dynamic responses to hepatic stresses in aging. In this collaborative proposal, empirical in vivo studies are combined with mathematical modeling and simulation to test effects of extrinsic and intrinsic factors on hepatocellular lineage dynamics and how these change as a part of the aging process in a genetically tractable animal model. What is known. Unlike most differentiated cell types, hepatocytes can proliferate. When normal liver cells are transplanted into mice having a genetic defect that autonomously compromises the endogenous hepatocytes, the grafted cells can complete >18 consecutive replicative cycles, resulting in full replacement of the endogenous hepatocytes and reconstitution of the liver with healthy cells. Using serial reconstitution through 7 consecutive recipient mice, a classic study showed that adult wild-type liver cells could undergo an average of at least 69 consecutive divisions 1. Thus, liver cells have a nearly unlimited capacity to proliferate 1 and may be "stem cell-like" in their regenerative immortality 1,2. Hepatocytes are also one of few cell types that undergo endoreplication and acytokinetic mitosis, resulting in polyploid nuclei and bi-nucleate cells, respectively. Most hepatocytes are polyploid and both diploid and polyploid hepatocytes can proliferate. Indeed, another study showed that the most active liver cell types for reconstituting compromised liver are polyploid hepatocytes 3. These background data indicate that: (1) liver cell populations may be infinitely proliferative; (2) hepatocytes are predisposed to becoming polyploid; and (3) polyploid hepatocytes are highly proliferative. Preliminary observations and the problem they reveal. We developed genetic marker systems for "time-stamping" hepatocyte lineages in vivo 4. In contrast to the prevailing model of the immortal hepatocyte, our systems show that hepatocyte lineages have both a finite half-life and a limited capacity to proliferate. We also developed a flow cytometry-based method of quantifying liver nuclei on the basis of ploidy and found that adult livers between 2- and 12-months of age exhibited nearly invariant ratios of diploid (2N), 4N, and 8N nuclei. Lastly, we developed a novel "ten-day chronometer" for newly differentiated hepatocyte lineages that allows us to quantitatively assess the contributions of pre-hepatocytic stem cells to liver growth, regeneration, and maintenance 5. Using this chronometer, we found that normal adult liver is continuously gaining new diploid hepatocyte lineages. We believe these replace lineages that die-off due to age or stress. Based on our observations, we suspect that only pre-hepatocyte cell types, not differentiated hepatocytes, have unlimited proliferative potential and that this rare population of cells underlies the proliferative immortality of liver. Our hypothesis and how we will test it. Based on our findings, we hypothesize that hepatocytes have a life history that includes birth from stem cells, age-related deterioration, and death. We predict that hepatic stresses, replication, ploidy, aneuploidy, and time will affect hepatocyte lineage life history dynamics. Moreover, the process of aging of the host animal might influence the life history dynamics of hepatocyte lineages. The quality of either the hepatic stem cells or the "liver niche" could change as livers age, resulting in differences in hepatocyte lineage birth rates, longevities, proliferative potentials, and stress resistance. To test our hypothesis, we will fulfill four aims: (1) Define birth-rates and longevities of hepatocyte lineages under normal and stressed conditions. (2) Determine what factors limit lineage longevity. (3) Measure the dynamic lineage-aging process in hepatocyte nuclei. (4) Examine how an animal's age and exposure-history affects the life history dynamics of hepatocyte lineages. Implications for human health in aging. Hepatocytes are generally thought to have a stem cell-like capacity to proliferate and regenerate lost or damaged liver tissue. Indeed, the term 'stem cell-like' invokes a level of immortality that has been tested in only a small number of situations. Clearly mouse ES cells, for example those we used to make our various lines of mice having targeted mutations, have been verified through years of culture and mouse-production as being indefinitely self-renewing; but is this true of all organ-specific stem cells? Maintenance of the self-renewing capacity of ES cells in culture requires a very strict environment or "niche" (e.g., media, supplements, attachment factors, feeder-cells, pH, etc.), so it may be reasonable to predict that the "quality" of organ-specific stem cells in vivo could also be intimately dependent on the niche that the host-organ provides. This niche could change with an animal's age or exposure history, yet these possibilities have not been previously considered. Here we propose an investigation into the aging process in differentiated hepatocyte lineages and how both this process and the contributions of hepatic stem cells change as animals age. Upon completion of this project, we will have: (a) developed and publicly disseminated novel mouse models for studying aging of hepatocyte lineages; (b) defined rates of birth and death of hepatocyte lineages under normal and several stressed states; (c) characterized the aging process in normal and stressed hepatocyte lineages; and (d) investigated how these processes change as a function of aging and exposure history of the host animal.

描述（由申请人提供）：我们开发了用于研究体内肝细胞谱系生命史动态的系统。我们建议定义决定肝细胞谱系出生率和寿命的因素，并描述它们对衰老过程中肝应激的动态反应。在这项合作提案中，体内实证研究与数学建模和模拟相结合，以测试外在和内在因素对肝细胞谱系动力学的影响，以及这些因素如何在遗传易处理的动物模型中作为衰老过程的一部分发生变化。已知什么。与大多数分化细胞类型不同，肝细胞可以增殖。当正常肝细胞被移植到具有自主损害内源性肝细胞的遗传缺陷的小鼠体内时，移植的细胞可以完成>18个连续复制周期，从而完全替代内源性肝细胞并用健康细胞重建肝脏。一项经典研究通过对 7 只连续受体小鼠进行连续重组，表明成年野生型肝细胞平均可以经历至少 69 次连续分裂1。因此，肝细胞具有几乎无限的增殖能力1，并且可能是“干细胞”。 -就像”他们的再生不朽1,2。肝细胞也是少数经历内复制和无细胞动力学有丝分裂的细胞类型之一，分别产生多倍体细胞核和双核细胞。大多数肝细胞是多倍体，二倍体和多倍体肝细胞都可以增殖。事实上，另一项研究表明，重建受损肝脏最活跃的肝细胞类型是多倍体肝细胞3。这些背景数据表明：（1）肝细胞群可能无限增殖； (2)肝细胞易于成为多倍体； (3)多倍体肝细胞具有高度增殖性。初步观察及其揭示的问题。我们开发了体内肝细胞谱系“时间戳”的遗传标记系统4。与普遍的永生肝细胞模型相比，我们的系统表明肝细胞谱系具有有限的半衰期和有限的增殖能力。我们还开发了一种基于流式细胞术的基于倍性的肝细胞核定量方法，发现 2 至 12 个月大的成年肝脏表现出几乎不变的二倍体 (2N)、4N 和 8N 细胞核比例。最后，我们为新分化的肝细胞谱系开发了一种新型“十天计时器”，使我们能够定量评估前肝细胞干细胞对肝脏生长、再生和维护的贡献5。使用这种计时器，我们发现正常成人肝脏不断获得新的二倍体肝细胞谱系。我们相信这些取代了因年龄或压力而消失的谱系。根据我们的观察，我们怀疑只有前肝细胞类型（而不是分化的肝细胞）具有无限的增殖潜力，并且这种罕见的细胞群是肝脏增殖永生的基础。我们的假设以及我们将如何测试它。根据我们的发现，我们假设肝细胞具有生命史，包括从干细胞诞生、与年龄相关的恶化和死亡。我们预测肝脏应激、复制、倍性、非整倍性和时间将影响肝细胞谱系生命史动态。此外，宿主动物的衰老过程可能会影响肝细胞谱系的生命史动态。肝干细胞或“肝脏生态位”的质量可能会随着肝脏年龄的增长而变化，导致肝细胞谱系出生率、寿命、增殖潜力和抗应激能力的差异。为了检验我们的假设，我们将实现四个目标：（1）定义正常和应激条件下肝细胞谱系的出生率和寿命。 (2) 确定哪些因素限制了谱系寿命。 (3)测量肝细胞核内的动态谱系老化过程。 (4) 检查动物的年龄和暴露史如何影响肝细胞谱系的生活史动态。老龄化对人类健康的影响。通常认为肝细胞具有类似干细胞的增殖和再生丢失或受损肝组织的能力。事实上，“类干细胞”一词所代表的永生水平仅在少数情况下得到了测试。显然，小鼠 ES 细胞，例如我们用来制造具有靶向突变的各种小鼠品系的细胞，经过多年的培养和小鼠生产已被证实具有无限的自我更新能力；但所有器官特异性干细胞都是如此吗？在培养物中维持 ES 细胞的自我更新能力需要非常严格的环境或“利基”（例如培养基、补充剂、附着因子、饲养细胞、pH 等），因此可以合理地预测体内器官特异性干细胞的“质量”也可能密切依赖于宿主器官提供的生态位。这个生态位可能会随着动物的年龄或接触史而改变，但以前没有考虑过这些可能性。在这里，我们建议对分化的肝细胞谱系的衰老过程以及该过程和肝干细胞的贡献如何随着动物衰老而变化进行研究。该项目完成后，我们将：（a）开发并公开传播用于研究肝细胞谱系衰老的新型小鼠模型； (b) 正常和几种应激状态下肝细胞谱系的出生率和死亡率； (c) 表征正常和应激肝细胞谱系的衰老过程； (d) 研究这些过程如何随着宿主动物的衰老和暴露史而变化。