STEM CELL THERAPY FOR DISEASES OF BONE IN A MOUSE MODEL

本研究的重点是利用小鼠成骨不全模型(OIM)作为模型系统来评价骨髓间充质干细胞(BMSCs)植入和参与骨的修复和再生的潜力。小鼠有一种自然发生的突变，这种突变导致proA2(I)链不表达，从而导致Al(I)同源三聚体在组织中积累。小鼠表现出骨量减少、皮质变薄和容易骨折的特点，是评估骨髓间充质干细胞作为治疗遗传性和非遗传性骨疾病靶点的良好模型。最近由Horwitz等人进行的临床试验。在患有严重OI的儿童中使用全骨髓，表明BMSCs可能为O1提供治疗选择。因此，需要检验的假设是：来自正常供者的骨髓间充质干细胞 将小鼠全身或局部注射到同基因受体小鼠体内，将植入受体小鼠的骨骼中，合成真正的骨细胞外基质，并有助于宿主骨的结构完整性。这些假说将被用于验证以下特定目的：1)证实注入OIM小鼠体内的细胞将植入OIM小鼠体内及骨折部位；2)证实植入骨骼内的细胞可分化为成骨细胞并合成真正的骨细胞外基质；3)证实植入骨骼内的细胞有助于保持骨骼的结构完整性。至 为了达到上述目的，将从正常供体小鼠的股骨和胫骨中建立BMSCs，并在OIM小鼠体内输注前标记表达LacZ或GFP基因的逆转录病毒以帮助细胞追踪或不标记。通过跟踪标记基因在组织中的表达和荧光原位杂交(FISH)，将跟踪注入细胞的命运。体内移植细胞向成骨细胞的分化将通过骨钙素和标记基因的共定位以及原位杂交来确定。通过测定由1和2个杂三聚体组成的I型胶原的存在来分析输注的细胞合成真实的细胞外基质。的结构完整性 宿主骨，将通过组织光度法、交联法、胶原含量和骨密度来确定。这些研究可能会为基于骨髓间充质干细胞的遗传性和非遗传性骨疾病的治疗提供更好的方法。

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.