MECHANISMS OF SKELETO-HAEMATOPOIETIC DIFFERENTIATION

描述(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。这一更新的目的是通过在软骨内骨化(EO)过程中用骨和骨髓取代肥大的软骨来形成造血骨髓环境，从而为骨骼-造血联系提供机制基础。这一建议源于我们的转基因(TG)小鼠和空白小鼠的X胶原蛋白，一种在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受压、骨量减少、骨髓再生不全和血细胞分化改变。我们认为，X胶原蛋白提供了一个由蛋白多糖(PGs)/糖胺多糖(GAG)稳定的结构网络，并隔离了细胞因子。网络的破坏导致生长板成分的重新分布和细胞因子的失衡；后者可能导致血液缺陷。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、肥大的软骨细胞和成骨细胞共培养来鉴定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修复造血缺陷；2)通过免疫组织化学和电子显微镜鉴定生长板/骨髓连接中哪些PG/GAG发生了改变，通过亲和共电泳法检测它们是否与X胶原结合，并通过HSC和肥大的软骨细胞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)检测巨噬细胞细胞因子表达异常与骨骼造血表型的严重程度之间的关系，鉴定内毒素攻击在启动/调节免疫反应中的缺陷，通过印迹阵列确定异常细胞因子的表达，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；以及4)检测其他肥大软骨改变模型(Grg5缺失和Snell侏儒)是否也存在类似的细胞因子失调引起的造血缺陷。

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.