Glycemic derangement and osteogenic cells:A model of Premature skeletal aging

描述（由申请人提供）：髋部骨折是老龄化人口中严重发病率和死亡率的催化剂。矛盾的是，绝经后2型糖尿病（T2DM）妇女发生髋部骨折的风险更高，尽管其骨密度高于无糖尿病妇女。骨量和骨质之间的不一致与进行性血糖紊乱的病理生理机制尚不清楚。在老化的骨骼中，循环因子的改变会对骨骼微环境产生不利影响。常驻间充质干细胞（MSCs）是成骨细胞的前体。在衰老过程中，它们表现出增殖减少和优先分化为脂肪细胞而不是成骨细胞谱系。尽管T2DM和衰老有显著的生理重叠，但关于常见循环因子如何对糖尿病骨和间充质干细胞产生有害的局部影响的研究很少。核心假设是，随着血糖水平的逐渐紊乱，循环中存在的因素的改变会破坏骨微环境，导致间充质干细胞增殖和分化减少，基质矿化减少，抗氧化应激能力受损，这与骨骼过早衰老一致。这一假设将通过追求三个具体目标来检验：1)确定糖耐量正常（NGT）、糖耐量受损（IGT）或T2DM老年受试者血清对人骨髓间充质干细胞（hMSC）增殖的影响；2)量化NGT、IGT或T2DM老年受试者血清改变骨髓间充质干细胞成骨细胞分化和基质矿化的能力；3)检测与这些受试者血清培养的未分化骨髓间充质干细胞氧化应激指标的诱导作用。在所有实验中，来自年轻健康女性供者（30-45岁）或老年供者（60 - 75岁）的NGT、糖耐量受损（IGT）或T2DM（每组n=10）的人血清（HuS）将与已建立的hMSCs系一起孵育。为了实现前两个目标，BrdU掺入、TUNEL检测、成骨细胞和脂肪细胞谱系分配标记（qRT-PCR）和细胞化学染色将用于确定hMSC的增殖、分化和矿化。为了实现第三个目标，将使用荧光活化细胞分选（FACS）、qRT-PCR和生化分析来定量活性氧和抗氧化酶的表达和活性。这种方法是创新的，因为它1)建立在一个新的T2DM模型的基础上，该模型是骨骼过早衰老的促成因素，2)通过确定NGT、IGT和T2DM HuS中系统因子对骨髓微环境的重要组成部分MSC的影响来验证该模型。这项研究具有重要意义，因为它将加强对糖尿病骨骼过早衰老的全身途径的理解。对2型糖尿病患者骨骼脆性机制的进一步了解将有助于早期干预和有针对性的治疗，以预防未来骨折后的残疾、发病率和死亡率。

DESCRIPTION (provided by applicant): Hip fracture is a catalyst for profound morbidity and mortality in the aging population. Paradoxically, postmenopausal women with type 2 diabetes mellitus (T2DM) are at higher risk for hip fractures despite having higher bone mineral density than women without diabetes. The pathophysiology underlying this incongruity between bone quantity and quality with progressive glycemic derangement is unknown. In the aging skeleton, alterations in circulating factors have adverse effects on the bone microenvironment. Resident mesenchymal stem cells (MSCs) are the precursors to bone-building osteoblasts. In aging, they demonstrate decreased proliferation and preferential differentiation into adipocyte rather than osteoblast lineages. Despite the significant physiologic overlap in T2DM and aging, there has been little investigation into how common circulating factors have detrimental, local effects on diabetic bone and MSCs. The central hypothesis is that with progressive glycemic derangement, alterations in factors present in the circulation disrupt the bone microenvironment resulting in reduced MSC proliferation and differentiation, diminished matrix mineralization and impaired defense against oxidative stress consistent with premature skeletal aging. This hypothesis will be tested by pursuing three specific aims: 1) To determine the effect of serum from elderly subjects with either normal glucose tolerance (NGT), impaired glucose tolerance (IGT) or T2DM on human MSC (hMSC) proliferation, 2) To quantify the capacity of sera from elderly subjects with either NGT, IGT or T2DM to alter osteoblastic differentiation and matrix mineralization in hMSCs, and 3) To examine the induction of oxidative stress indices on undifferentiated hMSCs incubated with sera from these same subjects. For all experiments, human sera (HuS) from either young healthy female donors (30-45 years) or elderly donors (60 - 75 years) with NGT, impaired glucose tolerance (IGT) or T2DM (n=10 per group) will be incubated with an established line of hMSCs. To accomplish the first two aims, BrdU incorporation, TUNEL assay, osteoblast and adipocyte lineage allocation markers in quantitative real-time PCR (qRT-PCR) and cytochemical staining will be used to determine hMSC proliferation, differentiation and mineralization. To accomplish the third aim, reactive oxygen species and antioxidant enzyme expression and activity will be quantified using Fluorescence Activated Cell Sorting (FACS), qRT-PCR and biochemical assays. This approach is innovative because it 1) is founded on a novel model of T2DM as a precipitant of premature skeletal aging, and 2) tests this model by determining the effects of systemic factors in NGT, IGT and T2DM HuS on the MSC, an essential constituent of the marrow microenvironment. This research is significant because it will enhance understanding of the systemic pathways which prematurely age the diabetic skeleton. Greater knowledge of the mechanisms contributing to skeletal fragility in T2DM will translate into early interventions and targeted therapeutics to prevent future disability, morbidity and mortality following fracture. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because identifying the pathogenesis of skeletal failure in T2DM will speed the discovery of effective strategies for hip fracture prevention in the aging, diabetic population. This research is also expected to stimulate future exploration into the role of hormonal, metabolic and musculoskeletal dysregulation in the development of disability and frailty with age.