MECHANISMS OF SKELETO-HAEMATOPOIETIC DIFFERENTIATION

骨骼造血分化机制

• 批准号: 6776660
• 负责人: Olena Jacenko
• 金额: $ 43.62万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6776660
• 关键词: autoimmunity; bone development; bone marrow; bone marrow transplantation; cartilage development; cell differentiation; chondrocytes; collagen disorder; cytokine; electron microscopy; enzyme linked immunosorbent assay; genetically modified animals; hematopoiesis; hematopoietic stem cells; immunocytochemistry; immunosuppression; laboratory mouse; microarray technology; mixed tissue /cell culture; mucopolysaccharides; neutralizing antibody; osteoblasts; pathologic ossification; proteoglycan

DESCRIPTION (provided by applicant): Hematopoietic disorders such as bone marrow failure, immune dysfunction, and certain cancers, may involve a link between the endochondral skeleton and marrow. The objective of this renewal is to provide a mechanistic basis for the skeleto-hematopoietic link, by addressing how a hematopoietic marrow environment may form by replacement of hypertrophic cartilage by bone and marrow during endochondral ossification (EO). This proposal stems from our transgenic (Tg) and null mice for collagen X, a matrix protein expressed in hypertrophic cartilage prior to EO. Murine skeleto-hematopoietic defects include growth plate compressions, osteopenia, marrow aplasia, and altered blood cell differentiation. We propose that collagen X provides a structural network that is stabilized by proteoglycans (PGs)/glycosaminoglycans (GAGs), and sequesters cytokines. Network disruption causes redistribution of growth plate components and a cytokine imbalance; the latter may cause the hemato-poietic defects. To test this hypothesis we will: 1) Identify the defective cellular component in the marrow environment of collagen X mice by co-cultures of hematopoietic stern cells (HSCs) with stromal cells, hypertrophic chondrocytes, and osteoblasts, and test if marrow transplantation can rescue the hematopoietic defects; 2) Identify which PGs/GAGs are altered in growth plate/marrow junctions by immunohistochemistry and electron microscopy, test if they bind collagen X by affinity coelectrophoreisis, and assess the role of these interactions in hematopoiesis by co-cultures of HSCs and hypertrophic chondrocytes GAG biosynthesis modulators; 3) Examine the relationship between the severity of the skeleto-hematopoietic phenotype and dysregulated cytokine expression by macrophage cytokine production, identify the defect in immune response initiation/regulation by endotoxin challenge, ascertain dysregulated cytokines by blot arrays, identify the tissue source of aberrant cytokine production, and induce/block the disease phenotype by injection of cytokines or neutralizing antibodies; and 4) Test if other murine models with altered hypertrophic cartilage (Grg5 null & Snell dwarfs) have similar hematopoietic defects caused by cytokine dysregulation.

描述（由申请人提供）：造血系统疾病，如骨髓衰竭、免疫功能障碍和某些癌症，可能涉及软骨内骨骼和骨髓之间的联系。这种更新的目的是通过解决在软骨内骨化（EO）过程中，骨髓和骨髓如何替代肥大软骨形成造血骨髓环境，为骨骼-造血联系提供机制基础。该提议源于我们的胶原X转基因（Tg）和无效小鼠，胶原X是EO前在肥大软骨中表达的基质蛋白。小鼠骨骼造血缺陷包括生长板压缩、骨质减少、骨髓发育不全和血细胞分化改变。我们提出，胶原蛋白X提供了一个结构网络，是稳定的蛋白聚糖（PG）/糖胺聚糖（GAG），并螯合细胞因子。网络中断导致生长板成分的重新分配和细胞因子失衡;后者可能导致造血缺陷。为了验证这一假设，我们将：1）通过造血干细胞（HSCs）与基质细胞、肥大软骨细胞和成骨细胞的共培养来鉴定胶原X小鼠骨髓环境中的缺陷细胞成分，并测试骨髓移植是否可以挽救造血缺陷; 2）通过免疫组织化学和电子显微镜鉴定哪些PG/GAG在生长板/骨髓连接处发生改变，通过亲和层析测试它们是否结合胶原X，并通过HSC和肥大软骨细胞GAG生物合成调节剂的共培养来评估这些相互作用在造血中的作用; 3）通过巨噬细胞细胞因子产生检查骨骼造血表型的严重性与失调的细胞因子表达之间的关系，通过内毒素攻击鉴定免疫应答启动/调节中的缺陷，通过印迹阵列确定失调的细胞因子，鉴定异常细胞因子产生的组织来源，并通过注射细胞因子或中和抗体诱导/阻断疾病表型;和4）测试具有改变的肥大软骨（Grg 5 null & Snell dwarfs）的其它鼠模型是否具有由细胞因子失调引起的类似造血缺陷。