Proteome-wide Expression Study of Osteogenic Cells

骨质疏松症是一个主要的公共卫生问题，特别是在妇女中。主要表现为低骨量 矿物质密度（BMD）。BMD具有> 60%的高遗传率。女性的BMD比男性低得多。我们和其他人 表明某些BMD基因/基因组区域具有性别特异性。更年期是一个最重要的 是女性生命中的生理事件，与女性特有的快速骨丢失有关。 骨髓间充质干细胞（BMMSC）和外周血单核细胞（PBM）是造血干细胞的前体。 成骨细胞（骨形成细胞）和破骨细胞（骨吸收细胞）。 我们的假设是，女性BMMSC和PBM中蛋白质表达谱的变化是 女性BMD变化的机制，并与绝经有关。 我们的主要目标是鉴定在女性BMMSC和PBM中差异表达的蛋白质：1）与 高与低骨密度; 2）绝经前后，从而确定蛋白质（及其基因）与 女性骨质疏松症和绝经的BMMSCs和PBMs。在女性中鉴定出的重要蛋白质将是 在男性样本中检测。 连同项目2，我们将招募80名健康女性和80名年龄匹配的健康女性， 年龄在50-55岁的男性，包括40名低BMD受试者和40名高BMD受试者（年龄匹配人群的底部或顶部20%） 每种性别，均为高加索人。每个女性BMD组包括20名术前和20名年龄匹配的 绝经后妇女。我们将采集新鲜骨髓（我们已经发表了大量研究 经验）和来自每个受试者的外周血样品。BMMSC和PBM将被分离，并且同样 分成两等份，一份用于项目2，一份用于项目3。在这个项目3中，我们将提取总蛋白质， 来自分离的BMMSC和PBM的等分试样。蛋白质组学分析实验和分析将在 使用MD-nano-LC-MS/MS对雌性动物进行检测。将通过以下方法验证鉴别出的显著差异表达蛋白质： 用雌性样本进行蛋白质印迹，以确定其在雌性中的重要性。在女性中验证的重要蛋白质将 用雄性样品通过蛋白质印迹法检查性别特异性。 将交叉检查在白人中获得的结果，以确定中国样本的种族一般性/特异性。 所鉴定的蛋白质及其基因的分子和细胞功能研究将作为例证进行 在本SCOR的项目2中。 本研究的主要结果（特别是从PBM获得的结果）可用于设计常规的 诊断性蛋白抗体芯片和/或蛋白标记物用于女性骨质疏松症的预后。特别是 结果将被用来提供一些功能性证据，当结合DMA多态性数据， 项目1和DMA微阵列数据在项目2中的SCOR，以强大和有效地识别基因和一些 对女性骨质疏松症的作用。

Osteoporosis is a major public health problem, especially in women. It is mainly characterized by low bone mineral density (BMD). BMD has high heritability of > 60%. Women have much lower BMD than men. We and others demonstrated that some BMD genes/genomic regions are gender specific. Menopause is a most significant physiology event in female's life and is associated with female-specific rapid bone loss. Bone marrow mesenchymal stem cells (BMMSCs) and peripheral blood monocytes (PBMs), are precursors for osteoblasts (bone formation cells) and osteoclasts (bone resorption cells), respectively. Our hypothesis is that changes in the protein expression profiles in female BMMSCs and PBMs underlie mechanisms of female BMD variation and are associated with menopause. Our major goals here are to identify proteins differentially expressed in BMMSCs and PBMs in women: 1) with high vs. low BMD; 2) before and after menopause, and thus identify proteins (and their genes) associated with female osteoporosis and menopause in BMMSCs and PBMs. The proteins identified significant in females will be examined in male samples. Together with Project 2, we will recruit 80 otherwise healthy females and 80 age-matched otherwise healthy males aged 50-55, including 40 subjects with low and 40 with high BMD (age matched population bottom or top 20% respectively) for each sex, all Caucasian. Each female BMD group includes 20 pre- and 20 age matched post-menopausal women. We will take fresh bone marrow (for which we have had extensive published research experience) and peripheral blood samples from each subject. BMMSCs and PBMs will be isolated and equally divided into two aliquots, one for Project 2 and one for Project 3. In this Project 3, we will extract the total proteins from aliquots of isolated BMMSCs and PBMs. Proteomic profiling experiments and analyses will be performed on Females using MD-nano-LC-MS/MS. Significant differentially expressed proteins identified will be verified by Western blotting with female samples for their importance in females. Significant proteins verified in females will be examined by Western blotting with male samples for sex-specificity. The results obtained in Caucasians will be cross checked for ethnic generality/specificity in Chinese samples. The molecular and cellular functional studies of the identified proteins and their genes will be pursued as exemplified in Project 2 of this SCOR. The major results (particularly those obtained from PBMs) of this study may be used to design customary diagnostic protein antibody chips and/or protein markers for prognosis of female osteoporosis. In particular, the results will be used to provide some functional evidence, when combined with the DMA polymorphism data in Project 1 and DMA microarray data in Project 2 of this SCOR to powerfully and efficiently identify genes and some of their functions for female osteoporosis.