MECHANISMS OF SKELETO-HEMATOPOIETIC DIFFERENTIATION

本研究旨在探讨软骨内骨化(EO)是否可能为血细胞分化建立必要的骨髓基质环境。这一假说源于我们对转基因(TG)和空白小鼠X胶原蛋白的分析，X胶原蛋白是一种在EO之前在肥大软骨中表达的基质蛋白。由此产生的小鼠骨骼造血缺陷包括生长板受压、骨量减少和骨髓再生障碍性贫血；后者会导致致命性或免疫力低下。我们的数据认为，X型胶原提供了一个加强生长板的结构框架，生长板的破坏改变了随后的骨和骨髓形成。这项建议将解决生长板中的结构缺陷是否会改变骨髓基质和随后的造血(目标1)，通过RT-PCR、Northern印迹、原位杂交、免疫组织化学和转基因来重新评估X胶原或转基因产物在骨骼外器官中的表达(目标1)。2)流式细胞术和免疫组织化学技术将识别受影响的血细胞谱系，并将这些缺陷的发病时间与骨骼改变联系起来。(目标2)。3)骨髓分析将涉及流式细胞术、免疫组织化学和细胞培养；4)骨髓移植将确定改变的造血是否源于基质或血祖细胞的缺陷；以及5)共培养分析将识别骨髓环境中的缺陷细胞成分(目标3)。6)弓形虫感染将测试动物的免疫反应，并将通过7)外周血液和骨髓分析脓毒症；8)ELIZA检测IL-12、干扰素-g和IL-10血清水平；9)注射细胞因子和中和抗体以解决骨髓再生不全的原因(目标4)来评估在TG和KO小鼠中看到的表型变异性。10)将通过将近亲交配的小鼠置于统一背景中来测试修饰基因的参与(目标4)。这些结果将为了解肥大的软骨和X胶原蛋白在骨骼造血发育中的作用提供依据。数据还可能建立软骨内骨骼生成和造血之间的机械联系，并为骨骼、免疫或转移受累的疾病的发病机制提供线索。

This proposal addresses whether endochondral ossification (EO), involving hypertrophic cartilage replacement by bone and marrow, may establish the marrow stromal environment prerequisite for blood cell differentiation. This hypothesis stems from our analyses of transgenic (Tg) and null mice for collagen X, a matrix protein expressed in hypertrophic cartilage prior to EO. Resultant murine skeleto-hematopoietic defects include growth plate compressions, osteopenia, and marrow aplasia; the latter results in lethality or compromised immunity. Our data maintain that collagen X provides a structural framework reinforcing growth plates, the disruption of which alters subsequent bone and marrow formation. This proposal will address if structural defects in growth plates alter the marrow stroma and subsequent hematopoiesis by l) re-evaluating collagen X or transgene product expression in extra-skeletal organs via RT-PCR, northern blot, in situ hybridization, immunohistochemistry, and transgenesis (Aim 1). 2) Flow cytometry and immunohistochemistry will identify affected blood cell lineages and correlate the temporal onset of these defects to the skeletal changes. (Aim 2). 3) Marrow analysis will involve flow cytometry, immunohistochemistry and cell culture; 4) marrow transplantation will establish if altered hematopoieis stems from defects in the Stroma or in blood progenitors; and 5) co-culture assays will identify the defective cellular component in the marrow milieu (Aim 3). 6) The animal's immune response will be tested by Toxoplasma gondii infections, and the phenotypic variability seen in both the Tg and KO mice will be assessed by 7) peripheral blood and marrow analysis for sepsis; 8) ELIZA for IL-12, IFN-g, and IL-10 serum levels; and 9) injections of cytokines and neutralizing antibodies will address the cause of marrow aplasia (Aim 4). 10) The involvement of a modifier gene will be tested by inbreeding mice into uniform backgrounds (Aim 4). Results will provide insights into hypertrophic cartilage and collagen X function in skeleto- hematopoietic development. Data may also establish mechanistic links between endochondral skeletogenesis and hematopoiesis, and provide clues into the pathogenesis of disorders with skeletal, immunologic, or metastatic involvement.