MECHANISMS OF MURINE CFU-E MATURATION

小鼠 CFU-E 成熟机制

• 批准号: 2016300
• 负责人: Terry Rogers Bishop
• 金额: $ 14.15万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-30 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2016300
• 关键词: antisense nucleic acid; apoptosis; complementary DNA; cytokine; erythroid stem cell; erythropoiesis; erythropoietin; gene expression; genetic library; growth factor receptors; high performance liquid chromatography; insulinlike growth factor; laboratory mouse; messenger RNA; northern blottings; nucleic acid sequence; oligonucleotides; polymerase chain reaction; protein kinase C; tissue /cell culture

We propose to elucidate mechanisms underlying maturation of erythroid colony-forming units (CFU-E) into red cells. Pursuing this goal, we use two populations of highly purified murine CFU-E: Generation I CFU-E forming 32-cell colonies, generation II forming 16-cell ones. For a rationale to dissect maturation, we draw upon our recent reports that (i) generation I CFU-E don't need erythropoietin (Epo) or other cytokines to progress to begins in generation II; (ii) DNA-breakdown characteristic of programmed cell death (apoptosis) first begins in generation II; (iii) Epo and insulin-like growth factor I (IGF-1) can then each almost completely forestall apoptosis; (iv) IGF-1 soon loses effectiveness whereas Epo remains effective until mid-maturation; (v) evident persistence of Epo action after it is withdrawn suggests that enduring transduction elements may have been unregulated by Epo; and (vi) for Epo- driven maturation to be optimal, a serum factor(s) other than IGF-1 is needed, especially when Epo levels are normal and this factor(s) then adds 30% or more to erythropoiesis. To identify factors (s) needed for optimal erythropoiesis, we apply reported candidates and serum fractions to well-defined serum-poor cultures to seek growth factors which can substitute for serum. We also seeks to learn why IGF-1 prematurely loses effectiveness and ask: It is due to loss of high-affinity receptors? In a parallel study, we better defined the time in CFU-E maturation when full-length Epo receptors (EpoR) capable of transducing prevention of apoptosis replace truncated EpoR which cannot. Results of such receptor analyses figure in our main purpose which is to identify molecular mechanisms by which apoptosis and cytokine response are transduced in CFU-E. Here we draw upon (i) genes whose expression in other tissues is associated with induction or prevention of apoptosis and (ii) gene expressed in CFU-E, especially those implicated by others in maturation and most especially those whose expression changes during maturation. Having established in preliminary studies that protein kinases (PK), particularly PKC, act in the pathway by which Epo forestalls CFU-E apoptosis, we focus on candidate PK whose changing transcription might underlie the temporal changes in apoptosis and/or cytokine response which we are observed. Drawing next upon pilot experiments which show that diverse PK clones can be isolated in varying recurrence frequencies from CFU-E RNA following reverse transcriptase polymerase-chain reactions primed with PK degenerate sequence oligomers, we plan to identify candidates PK stochastically. For example, using random PK clones developed from generation I CFU-E, from generation II plus Epo, from generation II plus IGF-1 and from generation II minus-cytokines, PK which recur unequally in the four CFU-E populations become candidates. Such PK, like non-PK candidates otherwise identified, will then be examined for maturation-dependent changes in RNA expression and, ultimately, for the influence their antisense oligomers have upon apoptosis and/or cytokine response. In these and other ways, we anticipate that elements underlying CFU-E maturation and apoptosis can be unabigously identified.

我们建议阐明红系成熟的机制。 集落形成单位(CFU-E)转化为红细胞。为了实现这一目标，我们使用 两个高纯度小鼠CFU-E群体：第一代CFU-E 形成32个细胞的集落，第二代形成16个细胞的集落。为. 剖析成熟的理论基础，我们借鉴了我们最近的报告，即(I) 第一代CFU-E不需要促红细胞生成素(EPO)或其他细胞因子 第二代开始的进展；(Ii)DNA-击穿特征 程序性细胞死亡(凋亡)第一次开始于第二代；(Iii) EPO和胰岛素样生长因子I(IGF-1)几乎可以各自 完全阻止细胞凋亡；(Iv)IGF-1即将失效 而促红细胞生成素直到成熟中期才有效；(V)明显 EPO在撤销后的持续行动表明，持久 转导元件可能不受EPO的调控；以及(Vi)对于EPO- 促成熟为最佳，胰岛素样生长因子-1以外的血清因子(S) 需要，特别是当促红细胞生成素水平正常的时候，这个因素(S) 增加30%或更多的红细胞生成。确定需要的因素(S) 最佳红细胞生成，我们应用已报道的候选和血清部分 到定义明确的血清贫乏培养，寻找可以 用来代替血清。我们还试图了解为什么IGF-1过早失利 问：这是由于高亲和力受体的丧失吗？在……里面 在一项平行研究中，我们更好地定义了CFU-E成熟的时间 全长EPO受体(EPOR)可转导预防 细胞凋亡取代了截短的EPOR，而EPOR不能。这种受体的结果 分析数字在我们的主要目的是鉴定分子 细胞凋亡和细胞因子反应的转导机制 CFU-E。在这里，我们利用(I)在其他组织中表达的基因 与诱导或防止细胞凋亡有关，以及(Ii)基因 在CFU-E中表达，尤其是在成熟过程中被其他人牵连的那些 尤其是那些在成熟过程中表达发生变化的基因。 在初步研究中确定了蛋白激酶(PK)， 尤其是PKC，在EPO阻止CFU-E的途径中发挥作用 细胞凋亡，我们关注候选的PK，它的转录变化可能 细胞凋亡和/或细胞因子反应的时间变化 我们被观察到了。接下来是试点试验，这些试验表明 可以在不同的重复频率中隔离不同的PK克隆 逆转录聚合酶链式反应检测CFU-E核糖核酸 用PK简并序列低聚物启动，我们计划鉴定 考生随机PK。例如，使用随机PK克隆 从第一代CFU-E发展而来，从第二代加上EPO发展起来，来自 第二代加IGF-1和来自第二代减去细胞因子，PK 在四个CFU-E人群中复发不均成为候选人。是这样的 PK，就像其他方式确定的非PK考生一样，然后将进行考试 对于依赖成熟的RNA表达的变化，最终对于 它们的反义寡聚物对细胞凋亡和/或 细胞因子反应。通过这些和其他方式，我们预计这些要素 潜在的CFU-E成熟和凋亡可以很容易地识别出来。