MECHANISMS OF SKELETO-HEMATOPOIETIC DIFFERENTIATION

骨骼-造血分化机制

• 批准号: 6517782
• 负责人: Olena Jacenko
• 金额: $ 31.58万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517782
• 关键词: RNase protection assay; Toxoplasma gondii; autoimmunity; bone development; bone marrow; cartilage development; cell differentiation; collagen; collagen disorder; cytokine; enzyme linked immunosorbent assay; flow cytometry; genetically modified animals; hematopoiesis; immunocytochemistry; immunosuppression; in situ hybridization; laboratory mouse; mixed tissue /cell culture; neutralizing antibody; normal ossification; northern blottings; pathologic ossification; polymerase chain reaction; tissue /cell culture

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.

该提议解决了涉及骨骼和骨髓肥厚软骨替代的内软骨软骨骨化（EO）是否可以建立骨髓基质环境的血细胞分化前提条件。该假设源于我们对胶原蛋白X的转基因（TG）和NULL小鼠的分析，胶原蛋白X是在EO之前在肥大软骨中表达的基质蛋白。由此产生的鼠骨架 - 毛刺性缺陷包括生长板压缩，骨质减少和骨髓aplasia；后者导致致死性或免疫力受损。 我们的数据坚持认为，胶原蛋白X提供了一个结构框架增强生长板，这种破坏会改变随后的骨骼和骨髓形成。该建议将解决生长板中的结构缺陷是否通过通过RT-PCR，Northern Blot，原位杂交，免疫组织化学化学和传输（AIL AIM 1）重新评估胶原蛋白X或转基因产物的表达是否会改变骨髓基质和随后的造血膜。 2）流式细胞仪和免疫组织化学将确定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。 （目标2）。 3）骨髓分析将涉及流式细胞仪，免疫组织化学和细胞培养； 4）骨髓移植将确定造血症是否因基质或血祖中的缺陷而改变； 5）共培养测定将确定骨髓环境中的缺陷细胞成分（AIM 3）。 6）动物的免疫反应将通过弓形虫的贡氏感染进行测试，TG和KO小鼠中看到的表型变异性将通过7）7）外周血和玛格分析来评估败血症； 8）IL-12，IFN-G和IL-10血清水平的Eliza；和9）注射细胞因子和中和抗体将解决骨髓aPlasia的原因（AIM 4）。 10）修饰基因的参与将通过近交小鼠进入统一背景来测试（AIM 4）。结果将为骨骼造血发育中肥厚软骨和胶原蛋白X功能提供见解。 数据还可以在内软骨骨骼生成和造血之间建立机械联系，并为骨骼，免疫或转移性参与的疾病发病机理提供线索。