MECHANISMS OF SKELETO-HEMATOPOIETIC DIFFERENTIATION

该建议涉及软骨内骨化（EO），包括由骨和骨髓替代肥大软骨，是否可以建立血细胞分化的骨髓基质环境先决条件。这一假设源于我们对转基因（Tg）和裸小鼠的胶原X（EO前肥大软骨中表达的基质蛋白）分析。由此产生的小鼠骨骼造血缺陷包括生长板压缩、骨质减少和骨髓发育不全;后者导致致死或免疫力低下。 我们的数据表明，胶原蛋白X提供了一个结构框架，加强生长板，破坏它改变了随后的骨和骨髓形成。该提案将通过以下方式解决生长板中的结构缺陷是否改变骨髓基质和随后的造血：1）通过RT-PCR、北方印迹、原位杂交、免疫组织化学和转基因重新评估骨外器官中的胶原X或转基因产物表达（目的1）。2)流式细胞术和免疫组织化学将识别受影响的血细胞谱系，并将这些缺陷的时间发作与骨骼变化相关联。 (Aim 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液祖细胞中的缺陷;和5）共培养测定将鉴定骨髓环境中的缺陷细胞组分（目的3）。6)将通过刚地弓形虫感染测试动物的免疫应答，并且将通过7）败血症的外周血和骨髓分析; 8）IL-12、IFN-g和IL-10血清水平的ELIZA;和9）细胞因子和中和抗体的注射来评估在Tg和KO小鼠中观察到的表型变异性，以解决骨髓发育不全的原因（目的4）。10)修饰基因的参与将通过将小鼠近亲繁殖到统一的背景中来测试（目标4）。结果将提供深入了解肥大软骨和胶原X功能的骨骼造血发育。 这些数据也可能建立软骨内骨骼发生和造血之间的机制联系，并为骨骼、免疫或转移性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.