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群中严重发病率和死亡率的催化剂。 奇怪的是，患有2型糖尿病（T2 DM）的绝经后女性尽管骨密度高于非糖尿病女性，但髋部骨折的风险更高。 骨质量和骨量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，循环因子的改变对骨微环境有不利影响。常驻间充质干细胞（MSC）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T2 DM和衰老的生理学重叠显著，但很少有研究表明常见的循环因子对糖尿病骨和MSC具有有害的局部影响。 中心假设是，随着进行性血糖紊乱，循环中存在的因子的改变破坏了骨微环境，导致MSC增殖和分化减少，基质矿化减少，对氧化应激的防御受损，与过早的骨骼衰老一致。 将通过追求三个具体目标来检验这一假设：1）确定来自具有正常葡萄糖耐量（NGT）、受损葡萄糖耐量（IGT）或T2 DM的老年受试者的血清对人MSC（hMSC）增殖的影响，2）定量来自具有NGT、IGT或T2 DM的老年受试者的血清改变hMSC中的成骨细胞分化和基质矿化的能力，和3）检测用来自这些相同受试者的血清孵育的未分化hMSC的氧化应激指数的诱导。对于所有实验，将来自年轻健康女性供体（30-45岁）或患有NGT、葡萄糖耐量受损（IGT）或T2 DM的老年供体（60 - 75岁）的人血清（HuS）（每组n=10）与已建立的hMSC系一起孵育。 为了实现前两个目标，将使用BrdU掺入、TUNEL测定、定量实时PCR（qRT-PCR）中的成骨细胞和脂肪细胞谱系分配标志物以及细胞化学染色来确定hMSC增殖、分化和矿化。 为了实现第三个目的，将使用荧光激活细胞分选（FACS）、qRT-PCR和生物化学测定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T2 DM作为过早骨骼衰老的促发剂的新模型上，和2）通过确定NGT、IGT和T2 DM HuS中的全身性因素对MSC（骨髓微环境的重要成分）的影响来测试该模型。 这项研究意义重大，因为它将增强对导致糖尿病骨骼过早老化的系统途径的理解。 对T2 DM骨骼脆弱性机制的更多了解将转化为早期干预和靶向治疗，以预防骨折后的未来残疾、发病率和死亡率。 公共卫生相关性：拟议的研究与公共卫生有关，因为确定T2 DM骨骼衰竭的发病机制将加速发现老年糖尿病人群髋部骨折预防的有效策略。这项研究也有望刺激未来探索激素，代谢和肌肉骨骼失调在残疾和脆弱的发展与年龄的作用。

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.