绝经后妇女骨质疏松性骨折相关蛋白的鉴定及其功能研究

Osteoporosis is a serious health problem in elderly females. It is characterized by low bone mineral density and high risk to osteoporotic fracture (OF). OF at hip is the most common type of OF, and has a mortality rate of 17% in females. OF has strong genetic determination. However, genes and proteins underlying OF have yet to be identified. Peripheral blood monocytes (PBM) can serve as procursors of osteoclasts and participate in bone metabolism in humans. Whether protein expression in PBM is related to OF has not been reported. Quantitative proteomics technologies can be used to identify and quantify thousands of proteins in a complex biological sample. However, application of this modern technology is rare in bone research field. In this project, we plan to recruit 80 Chinese postmenopausal women, including 40 patients with hip OF and 40 healthy controls without any fracture history. We will isolate PBM and extract proteins. We will utilize the liquid chromatography and masss spectrometry technologies to dissect PBM proteome-wide protein expression profile for each subject, and identify differentially expressed proteins (DEPs) between the fracture vs. non-fracture samples. Based on the findings, we will conduct Western blotting to validate the DEPs, and perform in vitro cell-based studies to explore the relationship of these DEPs with monocyte migration, osteoclast differentiation, and osteoclast bone-resorption activity. Through these studies, we will ascertain whether these protein(s) regulate osteoclastogenesis and bone resorption. After accomplishment of this project, we expect to identify signifiant proteins related to OF and characterize their functional mechanism in humans, hence to provide new molecular targets for osteoporosis prevention, intervention, and treatment.

骨质疏松症严重危害老年妇女健康，主要表现为低骨密度和骨质疏松性骨折（OF）。髋部OF是最常见的OF类型，致死率高达17%。OF具有很强的遗传决定性，与其相关基因和蛋白还有待鉴定。外周血单核细胞是破骨细胞前体细胞，参与骨代谢。单核细胞的蛋白质表达是否与骨折有关目前尚无报道。定量蛋白质组学技术可同时鉴定和定量复杂生物样品中成千上万个蛋白质。该技术在骨学研究领域的应用较少。本项目将征集80个绝经后妇女（40个髋部OF，40个健康对照），利用液相色谱和质谱技术分析每个受试者的血液单核细胞蛋白质表达谱，鉴定两组样本间显著差异表达的蛋白质。在此基础上，将通过Western印迹验证差异表达的蛋白，并通过体外细胞实验探索这些蛋白质与单核细胞的迁移、破骨细胞的分化形成、和破骨活性的关系，从而确定这些蛋白质是否调节破骨生成和骨吸收。本项目可望鉴定OF相关蛋白并揭示的作用机制，为妇女骨质疏松的预治提供新的靶标。

骨质疏松症严重危害老年人健康，特别是绝经后妇女。骨质疏松症主要表现为低骨密度和易发生骨质疏松性骨折（OF）。髋部OF常见且致死率高。OF受遗传因素影响，其致病基因及蛋白有待鉴定，具有临床应用价值的生物标志物尚待研发。外周血单核细胞是破骨细胞前体细胞、参与骨代谢。单核细胞所表达的蛋白质是否与OF有关尚无报道。..本项目共征集两组中国汉族老人(N>80)，包括51例绝经妇女（髋部OF27例，对照24例）和36例男性（髋骨OF18例，对照18例）。采集了5ml静脉血用于单核细胞分离、细胞总蛋白提取。利用非标记定量蛋白质组学方法和液相色谱及质谱技术解析了病例组和对照组的细胞蛋白质组表达谱，共鉴定和定量了约三千蛋白质。通过统计学分析发现约三百个蛋白质的细胞表达水平在女性病例和对照间存在显著差异，其中72个蛋白质在男性中也存在相同趋势的变化、且变化显著。通过生物信息学分析揭示了这些蛋白质区分病例和对照的能力，构建了骨折相关蛋白质的相互作用网络并揭示了重要节点蛋白，揭示了这些骨折相关蛋白质行使的分子功能、参与的生物学过程。通过Western印迹实验对关键蛋白质的组间差异进行了验证，通过体外细胞学实验进一步检测了其对成骨细胞及单核-破骨细胞功能的影响。..通过实施本项目，项目组发现了一批与中国汉族老人骨质疏松性骨折相关的蛋白质并揭示了关键蛋白质的骨细胞生物学作用机制。如：某蛋白质A在骨折病人的单核细胞中仅低量表达，该蛋白质可分泌到胞外，其血浆浓度在OF病例中明显低于同龄健康对照组；该蛋白质在生理浓度下可促进成骨、并具有剂量效应。在骨密度高的老年人中，该蛋白质的单核细胞表达量及血浆含量都显著高于骨密度低的同龄人或同龄OF病人。表明：单核细胞表达的蛋白质可能通过血循环调节成骨，其表达不足导致成骨不足、骨密度低、OF患病风险增加。..本项目所鉴定的功能性的骨折相关蛋白质为骨质疏松的早期诊断和干预提供了新的靶标，其转化应用价值有待在大样本及前瞻性队列中进一步评估。