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

描述(申请人提供)：髋部骨折是老年人口严重发病率和死亡率的催化剂。矛盾的是，绝经后患有2型糖尿病(T2 DM)的女性尽管比没有糖尿病的女性有更高的骨密度，但患髋部骨折的风险更高。导致进行性血糖紊乱的骨量和质量不一致的病理生理学机制尚不清楚。在老化的骨骼中，循环因子的变化会对骨骼微环境产生不利影响。间充质干细胞(MSCs)是成骨细胞的前体细胞。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。尽管在T2 DM和衰老方面有显著的生理重叠，但关于常见的循环因素如何对糖尿病骨骼和MSCs产生有害的局部影响的研究很少。中心假说是，随着进行性血糖紊乱，循环中存在的因素的改变扰乱了骨骼微环境，导致MSC增殖和分化减少，基质矿化减少，并与骨骼过早老化相一致的对氧化应激的防御能力减弱。这一假设将通过追求三个特定目标来验证：1)确定糖耐量正常(NGT)、糖耐量受损(IGT)或T2 DM老年患者的血清对人骨髓间充质干细胞(HMSC)增殖的影响；2)量化NGT、IGT或T2 DM老年患者血清改变hMSCs成骨分化和基质矿化的能力；3)检测氧化应激指数对未分化的hMSCs与这些受试者血清孵育的诱导作用。在所有实验中，来自患有NGT、糖耐量受损(IGT)或T2 DM(n=10)的年轻健康女性捐赠者(30-45岁)或老年捐赠者(60-75岁)的人血清(HUS)将与已建立的hMSCs系孵育。为了实现前两个目标，将采用BrdU掺入、TUNEL法、成骨细胞和脂肪细胞的实时定量聚合酶链式反应(qRT-PCR)标记和细胞化学染色来检测hMSC的增殖、分化和矿化。为了实现第三个目标，将使用荧光激活细胞分类(FACS)、qRT-PCR和生化分析来定量检测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作为骨骼过早老化的沉淀剂的新模型上，2)通过确定NGT、IGT和T2 DM HUS中的系统因素对骨髓微环境的重要组成部分MSC的影响来测试该模型。这项研究意义重大，因为它将加强对糖尿病骨骼过早老化的系统途径的理解。更多地了解导致2型糖尿病骨骼脆弱的机制将转化为早期干预和有针对性的治疗，以防止未来骨折后的残疾、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.