The Effects of CFTR Dysfunction on Bone Formation

描述(由申请人提供)：囊性纤维症(CF)患者的中位生存期已增加到35岁。随着越来越多的CF患者能够很好地存活到成年，骨质量差已经成为这种慢性疾病的一个具有挑战性的负担。低骨密度和高骨折率在CF中有很好的记录，但了解较少。尽管囊性纤维性骨病(CFBD)在成人中的患病率很高，但它也存在于儿童中。尽管它的流行，CFBD仍然是最小的特点，对其发病机制的了解仍然有限。我们的主要目标是在结构和细胞水平上全面描述囊性纤维化小鼠模型中的CFBD，因为它有可能提供对CFBD发病机制的更深层次的了解。由于这些动物没有严重的肺部或胰腺疾病，因此小鼠CF模型对于评估CFTR在骨骼动态平衡中的直接作用特别有用。初步数据表明，从3周大到成年，这些CFTR-/-动物的骨量减少，其特征是骨小梁变薄，皮质骨变薄，骨体积和强度降低，从而提供了类似人类疾病的模型。我们假设，这种CF模型将表现出与在CF患者中观察到的相似的复杂的骨损害，具有低骨密度和增加的骨脆性。很可能在人类的CFBD中，许多骨细胞系中CFTR功能的改变起着核心作用。然而，根据我们最初的研究，我们认为成骨细胞中CFTR的缺乏可能是破骨细胞功能改变的主要问题，而破骨细胞功能改变是炎症的次要后果。通过使用原位杂交、RT-PCR和免疫组织化学以及标准膜片钳技术，将检测CFTR在成骨细胞和破骨细胞中的功能作用。此外，成骨细胞和破骨细胞将在体外进行检查，以确定哪种细胞类型对原发损害负责。最后，我们将使用重复性雾化脂多糖模型，通过使用静态和动态骨组织形态计量学和血清骨转换标志物来定量骨密度和微结构细胞活动，以确定慢性炎症是否增加了小鼠CFBD模型的骨骼表型。通过阐明低骨密度的确切性质和演变，以及阐明骨细胞活动是否存在原发缺陷，可以进行更多的循证干预试验来解决CFBD的预防和治疗问题。有了更强壮、更健康的骨骼，CF患者的生活质量可以得到改善。 公共卫生相关性：这项研究项目旨在从结构和细胞水平全面描述囊性纤维化小鼠模型中的囊性纤维性骨病(CFBD)，有可能为CFBD的发病机制提供更深入的了解。通过阐明低骨量密度的确切性质和演变以及成骨细胞和破骨细胞在疾病过程中的作用，可以进行更有针对性的干预试验来预防和处理CFBD。认为成骨细胞或破骨细胞中功能CFTR的缺失直接导致了所观察到的骨病，这是当前CF相关骨病范式的一种转变。

DESCRIPTION (provided by applicant): Median survival for people with Cystic Fibrosis (CF) has increased to >35 years of age. As more CF patients survive well into adulthood poor bone quality has emerged as a challenging burden of this chronic disease. Low bone density and increased fracture rates in CF are well-documented but poorly understood. Although the prevalence for CFBD (Cystic Fibrosis bone disease) is high in adults, it is also present in children. Despite its prevalence, CFBD remains minimally characterized and an understanding of its pathogenesis remains limited. Our major objective is to fully characterize CFBD in a murine model of cystic fibrosis at the structural and cellular level, as it has the potential to provide a deeper understanding of the pathogenesis of CFBD. Murine models of CF are particularly useful for assessing the direct role of CFTR in skeletal homeostasis as these animals do not have severe pulmonary or pancreatic disease. Preliminary data generated demonstrate that from as early as 3 weeks of age through adulthood, these cftr-/- animals have decreased bone mass which is characterized by thinner trabeculae, and thinner cortical bone, as well as, decreased bone volume and strength thus providing a model similar to human disease. We hypothesize that this CF model will manifest a complex bone lesion with low bone density and increased bone fragility similar to that which is observed in CF patients. It is likely that altered CFTR function in a number of bone cell lineages plays a central role in CFBD in humans. However, based on our initial studies we believe the lack of CFTR in the osteoblast is likely to be the primary problem with alterations in osteoclast function a secondary consequence of inflammation. By employing in situ hybridization, RT-PCR, and immunohistochemistry as well as standard patch clamp techniques, the functional role of CFTR in osteoblasts and osteoclasts will be examined. In addition osteoblasts and osteoclasts will be examined in vitro to determine which cell type is responsible for the primary lesion. Lastly we will use a repetitive aerosolized LPS model to determine if chronic inflammation augments the skeletal phenotype of CFBD in the murine model by quantifying bone density and micro-architecture cellular activity using static and dynamic bone histomorphometry and serum markers of bone turnover. By clarifying the exact nature and evolution of the low BMD, and elucidating whether there is a primary defect in bone cell activity, more evidence-based intervention trials can be undertaken to address prevention and management of CFBD. With a stronger, healthier skeleton, the quality of life of patients with CF can be improved. PUBLIC HEALTH RELEVANCE: This research project, which aims to fully characterize cystic fibrosis bone disease (CFBD) in murine models of cystic fibrosis at the structural and cellular levels, has the potential to provide a deeper understanding of the pathogenesis of CFBD. By clarifying the exact nature and evolution of the low bone mass density and elucidating the contribution of the osteoblast and osteoclast to the disease process, more focused intervention trials can be done to address prevention and management of CFBD. The idea that the lack of functionally CFTR in either the osteoblast or osteoclast contributes directly to the observed bone disease is a shift in the current CF related bone disease paradigm.