MECHANISMS OF SKELETO-HEMATOPOIETIC DIFFERENTIATION

本研究旨在探讨软骨内成骨（EO）是否为血细胞分化提供了骨髓基质环境。这一假设源于我们对转基因（Tg）和无胶原X小鼠的分析，胶原X是在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压迫、骨质减少和骨髓发育不全；后者导致致命或免疫力受损。我们的数据表明，X胶原蛋白提供了一个结构框架来加强生长板，其破坏改变了随后的骨骼和骨髓形成。该提案将通过以下方法解决生长板结构缺陷是否会改变骨髓基质和随后的造血：1)通过RT-PCR、northern blot、原位杂交、免疫组织化学和转基因（目的1）重新评估骨骼外器官中X胶原或转基因产物的表达（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病与骨骼变化联系起来。（目标2）。3)骨髓分析将包括流式细胞术、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源于基质缺陷还是造血祖细胞缺陷；5)共培养试验将识别骨髓环境中有缺陷的细胞成分（目标3）。6)动物的免疫反应将通过弓形虫感染进行测试，Tg和KO小鼠的表型变异性将通过外周血和骨髓分析来评估败血症；8) elisa检测血清IL-12、IFN-g、IL-10水平；9)注射细胞因子和中和抗体将解决骨髓发育不全的原因（目标4）。10)修饰基因的参与将通过在统一背景下近亲繁殖的小鼠进行测试（目标4）。结果将为肥大软骨和X胶原蛋白在骨骼造血发育中的功能提供见解。数据还可以建立软骨内骨骼形成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.