INHIBITED INTRAMEMBRANEOUS BONE HEALING IN DIABETES

描述(由申请人提供)：糖尿病并发症的部分原因是血糖水平升高。这会导致蛋白质的非酶糖基化，形成高级糖基化终末产物(AGE)。年龄在糖尿病的许多并发症中起着重要作用。骨量减少、骨密度低、骨质脆弱是1型糖尿病的并发症，被称为“糖尿病骨病”。令人惊讶的是，关于AGE在调节骨愈合、骨形成和成骨细胞功能中的作用，人们知之甚少。例如，晚期糖基化终产物受体(RAGE)在成骨细胞中的存在和活性尚未见报道。这项研究的主要假设是，AGE通过结合和激活成骨细胞中的RAGE来抑制骨愈合和形成。我们认为，这导致了NFKB的激活以及关键的成骨细胞生长因子和细胞外基质基因的转录抑制和下调。 目的1在体内检测糖尿病和非糖尿病小鼠颅骨缺损愈合过程中生长因子和细胞外基质产物(BMP-1、BMP-2、BMP-4和I型胶原)的表达。多个小剂量链脲佐菌素方案将在Balb/c小鼠中诱导糖尿病；选定的研究将在非化学诱导的小鼠糖尿病模型(NOD株)中进行。糖尿病动物骨愈合的抑制程度和RAGE的表达模式将通过定量组织形态计量学和定量免疫组织化学测量来确定。研究将通过局部应用AGE在非糖尿病小鼠的颅骨缺损中直接确定AGE在减少糖尿病骨形成中的作用。这种模拟糖尿病骨的程度将通过测量对愈合的抑制和对相同的生长因子和细胞外基质产物的调节来确定。 目的2将在原代培养的大鼠成骨细胞中确定AGE通过RAGE激活的核因子-KB抑制成骨细胞生长因子和I型胶原的产生。RAGE功能阻断抗体的研究将确定特定成骨细胞基因调控中依赖于年龄/RAGE的核因子-KB的激活机制。核因子-KB的激活在下调成骨细胞靶基因中的作用将直接通过转染超抑制因子32A/36A-IKB-α来确定，后者是一种有效和特异的核因子-KB激活抑制物。这些研究将确定导致糖尿病骨疾病的新机制；并应导致为这种日益普遍的临床疾病确定新的治疗靶点。

DESCRIPTION (provided by applicant): Complications of diabetes result in part from elevated serum glucose levels. This leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGE's). AGE's play a significant role in many complications of diabetes. Osteopenia and low mineral density and weak bones is a complication of Type 1 diabetes and is known as "diabetic bone disease". Surprisingly little is known regarding the role of AGE's in modulating bone healing, bone formation, and osteoblast function. For example, the presence and activity of the receptor for advanced glycation end products (RAGE) has not been reported in osteoblasts. The principal hypothesis of the proposed research is that AGE's inhibit bone healing and formation by binding and activating RAGE in osteoblasts. We propose that this results in NFKB activation and transcriptional repression and down-regulation of key osteoblast growth factors and extracellular matrix genes. Aim 1 will measure in vivo the expression of selected growth factors and extracellular matrix products (BMP-1, BMP- 2, BMP-4, and type I collagen) in healing calvaria defects made in diabetic and non-diabetic mice. Diabetes will be induced by the multiple low dose streptozotocin protocol in Balb/c mice; selected studies will be performed in the nonchemically-induced murine diabetic model (NOD strain). The degree of inhibition of bone healing in diabetic animals and expression patterns of RAGE will be determined by quantitative histomorphometric and quantitative immunohistochemical measurements. Studies will directly determine the role of AGE's in diminished diabetic bone formation by local application of AGE's to calvaria defects in non-diabetic mice. The degree to which this mimics diabetic bone will be determined by measuring inhibition of healing and regulation of the same growth factors and extracellular matrix products. Aim 2 will determine in vitro in primary rat osteoblast cell cultures that AGE's inhibit production of osteoblast growth factors and type I collagen via RAGE activated NF-KB. RAGE function blocking antibody studies will identify the AGE/RAGE-dependent NF-KB activation mechanism in the regulation of the specified osteoblast genes. The role of NF-KB activation in down-regulating target osteoblast genes will be directly determined by transfection with the super-repressor 32A/36A IKB-alpha, a potent and specific inhibitor of NF-KB activation. These studies will identify a new mechanism that contributes to diabetic bone disease; and should lead to the identification of new therapeutic treatment targets for this increasingly prevalent clinical condition.