MECHANISMS OF RETINOID EFFECTS ON SKELETOGENESIS

类维生素A影响骨骼形成的机制

• 批准号: 6107419
• 负责人: DOUGLAS Frank PAULSEN
• 金额: $ 9.59万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107419
• 关键词: biological signal transduction; chick embryo; ectoderm; enzyme linked immunosorbent assay; fibroblast growth factor; growth factor receptors; high performance liquid chromatography; in situ hybridization; limb regeneration; mesenchyme; musculoskeletal regeneration; northern blottings; nutrition related tag; polymerase chain reaction; receptor expression; retinoate; retinoid binding proteins; scintillation counter; tissue /cell culture; vitamin metabolism; vitamin receptor; western blottings

Limb defects afflict more than one out of every 2,000 live human births. The mechanisms underlying these often devastating defects are unclear. A fundamental aspect of limb development is outgrowth. The project's long term objective is to understand the regulation of this process. The immediate goal is to test the hypothesis that the ability of the limb bud's apical ectodermal ridge (AER) to promote limb outgrowth involves an ability to maintain cellular retinoic acid-binding protein (CRABP) expression in sub-ridge limb mesenchyme and thereby to limit the retinoic acid (RA) encountered by nuclear RA receptors in these cells. Corollary hypothesis to be tested are 1) than an AER signal maintains CRABP expression, 2) that the AER signal is FGF-8, 3) that CRABP promotes RA degradation, 4) that RA degradation prevents stem-cell differentiation, 5) that preventing stem-cell differentiation prevents FGF-receptor (FGFR) isoform switching, and 6) that preventing FGF-R isoform switching maintains limb mesenchymal competence to respond to the AER. Six specific aims are proposed to test this hypothesis. Aim 1 is to document AER effects on CRABP expression in the subjacent limb-bud mesenchyme in vivo. Two approaches will be taken. CRABP expression will be examined in chick limbs whose AERs have been excised at different stages of limb outgrowth, and limbs into which extra AERs have been grafted. Unoperated contralateral limbs will serve as controls. Aim 2 is to determine the AER's effects on retinoic acid metabolism. Distal limb fragments, intact or with their AER's previously surgically removed, will be incubated in vitro with 2/H-RA. Incubated tissue and media extracts will be analyzed by HPLC to compare metabolite profiles produced by the different preparations. Aim 3 is to determine RA effects on CRABP expression. Serum- free micro-cultures of limb mesenchyme will be treated with all-trans-ERA, 9-cis-RA, or 3,4-didehydro-RA. CRABP protein levels will be assessed by in situ ELISA and Western blots of culture extracts. Aim 4 is to determine FGF effects on CRABP expression. Micro-cultures of mesenchyme from various limb regions grown in serum-free medium containing FGF-2, FGF-4, FGF-8, or FGF-10 will be subjected to whole-mount in situ hybridization using a digoxigenin-labeled CRABP riboprobe in the wells of the plate. Aim 5 is to determine CRABP's effect on responses to retinoic acid. Micro-cultures transiently transfected with a eukaryotic expression vector containing a 700 bp. CRABP cDNA construct, will be treated with all-trans, 9-cis-, or 3,4-didehydro-RA, and effects on growth, chondrogenesis, and FGFR expression will be measured. Aim 6 is to determine CRABP's effect on RA metabolism. Chick limb-bud mesenchymal cells will be transiently transfected with a eukaryotic expression vector containing a 700 bp CRABP cDNA. Transfected cells will be harvested, washed, suspended in serum-free medium, and incubated with RA radio-labeled to high specific activity.

每2 000个活产婴儿中就有一个以上患有肢体缺陷。 这些通常具有破坏性的缺陷背后的机制尚不清楚。一 肢体发育最基本的方面是向外生长。这个项目很长 我们的目标是了解这个过程的规则。的 当前的目标是检验肢体的能力 芽的顶端外胚层嵴（AER），以促进肢体生长涉及一个 维持细胞视黄酸结合蛋白的能力 在亚嵴肢间充质中表达，从而限制视黄质 酸（RA）遇到的核RA受体在这些细胞。推论 待检验假设是1）AER信号维持CRABP 表达，2）AER信号是FGF-8，3）CRABP促进RA 4）RA降解阻止干细胞分化，5） 阻止干细胞分化可以阻止FGF受体（FGFR） 同种型转换，和6）防止FGF-R同种型转换 维持肢体间充质对AER的反应能力。六项具体 目的是为了检验这一假设。目标1是记录AER 在体内对小腿下肢芽间充质中CRABP表达的影响。 将采取两种办法。将在鸡中检测CRABP表达 在肢体生长的不同阶段切除AER的肢体， 以及移植了额外AER的肢体未手术 对侧肢体作为对照。目标2是确定 AER对维甲酸代谢的影响。远端肢体碎片，完整 或者他们的AER已经被手术切除， 2/H-RA体外培养。孵育组织和培养基浸提液将由以下人员进行分析： HPLC比较不同方法产生的代谢产物谱 准备工作。目的3是确定RA对CRABP表达的影响。血清- 肢体间充质的游离微培养物将用全反式ERA处理， 9-顺式-RA或3，4-二脱氢-RA。CRABP蛋白水平将通过以下方法进行评估： 培养物提取物的原位ELISA和Western印迹。目标4：确定 FGF对CRABP表达的影响。不同来源间充质的微培养 在含有FGF-2、FGF-4、FGF-8或FGF-8的无血清培养基中生长的肢体区域， FGF-10将进行整体原位杂交， 洋地黄毒苷标记的CRABP核糖探针在板的威尔斯孔中。目标5： 确定CRABP对视黄酸反应的影响。微培养 瞬时转染了含有 700 bp。CRABP cDNA构建体将用全反式、9-顺式或 3，4-二脱氢-RA以及对生长、软骨形成和FGFR的影响 表达将被衡量。目的6：观察CRABP对类风湿关节炎的治疗作用 新陈代谢.鸡肢芽间充质细胞将短暂地 用含有700 bp CRABP的真核表达载体转染 cDNA。将转染的细胞收获、洗涤、悬浮在无血清培养基中， 培养基中，并与RA放射性标记的高比活性孵育。