STEM CELL THERAPY FOR DISEASES OF BONE IN A MOUSE MODEL

干细胞治疗小鼠模型中的骨疾病

• 批准号: 6889423
• 负责人: CHRISTOPHER NIYIBIZI
• 金额: $ 33.95万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-27 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6889423
• 关键词: SDS polyacrylamide gel electrophoresis; bone marrow; cell differentiation; disease /disorder model; enzyme linked immunosorbent assay; fluorescent in situ hybridization; genetically modified animals; green fluorescent proteins; high performance liquid chromatography; histology; immunofluorescence technique; laboratory mouse; morphometry; musculoskeletal disorder therapy; musculoskeletal regeneration; osteoblasts; osteocalcin; osteogenesis imperfecta; polymerase chain reaction; stem cell transplantation; tissue /cell culture

The focus of the present proposal is to utilize a mouse model of osteogenesis imperfecta (oim) as a model system to evaluate the potential of the bone marrow derived mesenchymal stem cells (BMSCs) to engraft and participate in repair and regeneration of bone. The mouse has a natural occurring mutation that results in non-expression of proa2(I) chains leading to the accumulation of al(I) homotrimers in tissues. The mouse exhibits osteopenia, cortical thinning and easy fracturing and is an excellent model for evaluating the potential of BMSCs as targets for the treatment of genetic and non-genetic diseases of bone. Recent clinical trial by Horwitz et al. using whole marrow in children with a severe form of OI, demonstrated that BMSCs may offer treatment options for O1. Therefore, the hypotheses to be tested are: BMSCs from normal donor mice administered systemically or locally into syngeneic recipient mice will engraft in the bones of the recipient mice, synthesize authentic bone extracellular matrix and contribute to the structural integrity of the host bone. The following specific aims will be used to test these hypotheses: 1) Demonstrate that the cells infused into oim mice will engraft in bone and in fracture sites created in oim mice 2) Demonstrate that the cells which engraft in bone differentiate into osteoblasts and synthesize the authentic bone extracellular matrix and 3) Demonstrate that the cells that engraft in bone contribute to the structural integrity of bone. To accomplish the above aims, BMSCs will be established from femurs and tibiae of normal donor mice and either marked with retroviruses expressing LacZ or GFP genes to aid in cell tracking or unmarked prior to infusion in oim mice. The fate of the infused cells will be tracked by following expression of the marker genes in tissue and by fluorescent in situ hybridization (fish). Differentiation of the transplanted cells into osteoblasts in vivo will be determined by co-localization of osteocalcin and marker genes and also by in situ hybridization. Synthesis of authentic extraceltular matrix by the infused cells will be analyzed by the determination of the presence of type I collagen comprised of 1 and 2 heterotrimers. Structural integrity of the host bone, will be determined by histophotometry, cross-linking, and collagen content and bone mineral density. The proposed studies may lead to the development of better treatments for genetic and non-genetic diseases of bone based on BMSCs.

本提案的重点是利用骨化不完美的小鼠模型（OIM）作为模型系统，以评估骨髓衍生的间充质干细胞（BMSC）的潜力，以植入并参与骨骼的修复和再生。小鼠具有天然发生的突变，导致proA2（i）链的不表达，导致组织中Al（i）同构元的积累。小鼠表现出骨质减少，皮质稀释和易于破裂，是评估BMSC作为治疗骨骼遗传和非遗传疾病的靶标的潜力的绝佳模型。 Horwitz等人最近的临床试验。在具有严重形式的OI的儿童中使用整个骨髓，证明BMSC可以为O1提供治疗选择。因此，要测试的假设是：来自正常供体的BMSC 全身或局部施用的小鼠将小鼠植入受体小鼠的骨骼中，合成正宗的骨外骨外基质，并有助于宿主骨的结构完整性。以下特定目的将用于检验这些假设：1）证明，注入OIM小鼠的细胞将植入骨骼和在OIM小鼠中产生的骨折部位中，2）证明，植入骨骼中的细胞分化为成骨细胞成骨细胞，并在骨外骨外基质和3）骨骼结构的骨骼结构的细胞中形成了骨外骨骼的骨骼。到 完成上述目的，将从正常供体小鼠的股骨和胫骨中建立BMSC，并用表达LACZ或GFP基因的逆转录病毒标记以帮助细胞跟踪或在OIM小鼠输注之前未标记。通过遵循组织中标记基因和荧光原位杂交（FISH）的表达来跟踪注入细胞的命运。将移植细胞分化为体内成骨细胞，将通过骨钙素和标记基因的共定位以及原位杂交来确定。通过确定由1和2个杂三聚体组成的I型胶原蛋白的存在，将分析由注入的细胞合成真实的脑外基质。结构完整性 宿主骨，将由组织粒测量法，交联，胶原蛋白含量和骨矿物质密度确定。拟议的研究可能导致基于BMSC的骨骼遗传和非遗传疾病的更好治疗方法。