MECHANISMS OF SKELETO-HAEMATOPOIETIC DIFFERENTIATION

描述（由申请人提供）：造血疾病，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。这项更新的目的是通过解决在软骨内成骨（EO）过程中骨质和骨髓如何替代肥厚软骨形成造血骨髓环境，为骨骼-造血联系提供机制基础。这一建议源于我们的转基因（Tg）和X胶原的小鼠，X胶原是在EO之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迫、骨质减少、骨髓发育不全和血细胞分化改变。我们提出胶原X提供了一个由蛋白聚糖(pg)/糖胺聚糖（GAGs）稳定的结构网络，并隔离细胞因子。网络破坏导致生长板成分重新分配和细胞因子失衡；后者可引起造血缺陷。为了验证这一假设，我们将：1)通过造血干细胞（hsc）与基质细胞、增生性软骨细胞和成骨细胞共培养，确定X胶原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生长板/骨髓连接处哪些pg /GAGs发生改变，通过亲和共电泳检测它们是否与X胶原结合，并通过hsc和肥大软骨细胞GAG生物合成调节剂共培养评估这些相互作用在造血中的作用；3)通过巨噬细胞产生细胞因子检测骨骼-造血表型的严重程度与细胞因子表达异常的关系，通过内毒素攻击确定免疫应答启动/调节缺陷，通过印迹阵列确定细胞因子表达异常，确定细胞因子产生异常的组织来源，通过注射细胞因子或中和抗体诱导/阻断疾病表型；4)检测其他增生性软骨改变的小鼠模型（Grg5 null & Snell dwarf）是否存在由细胞因子失调引起的类似造血缺陷。

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.