STEM CELL THERAPY FOR DISEASES OF BONE IN A MOUSE MODEL

本研究的重点是利用小鼠成骨细胞模型（oim）作为模型系统来评估骨髓间充质干细胞（BMSCs）移植并参与骨修复和再生的潜力。小鼠具有天然存在的突变，其导致proa 2（I）链的不表达，从而导致al（I）同源三聚体在组织中的积累。小鼠表现出骨质减少、皮质变薄和容易骨折，是评估BMSCs作为治疗遗传和非遗传性骨疾病的靶点的潜力的极好模型。Horwitz等人最近在患有严重OI的儿童中使用全骨髓进行的临床试验表明，BMSC可能为O 1提供治疗选择。因此，待检验的假设是：来自正常供体的BMSCs 将小鼠全身或局部给药到同系受体小鼠中，将移植到受体小鼠的骨中，合成真实的骨细胞外基质，并有助于宿主骨的结构完整性。以下具体目的将用于检验这些假设：1）证明输注到oim小鼠中的细胞将在oim小鼠中产生的骨和骨折部位中植入; 2）证明植入骨中的细胞分化成骨细胞并合成真实的骨细胞外基质; 3）证明植入骨中的细胞有助于骨的结构完整性。到 为了实现上述目的，将从正常供体小鼠的股骨和胫骨建立BMSC，并且用表达LacZ或GFP基因的逆转录病毒标记以帮助细胞追踪或在输注到oim小鼠中之前不标记。通过跟踪组织中标记基因的表达和荧光原位杂交（FISH）跟踪输注细胞的命运。将通过骨钙素和标记基因的共定位以及原位杂交来确定移植细胞在体内分化成成骨细胞。将通过测定由1和2个异源三聚体组成的I型胶原的存在来分析输注细胞合成真实的细胞外基质。结构完整性 宿主骨将通过组织光度法、交联、胶原含量和骨矿物质密度来确定。拟议的研究可能会导致更好的治疗遗传性和非遗传性骨疾病的基础上BMSCs的发展。

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.