TRANSGENIC OSTEOBLASTS TO EXAMINE ECM FUNCTIONS

这项提议的总体目标是开发新的实验 阐明骨基质分子功能作用的策略， 骨钙素(OC)和骨涎蛋白(BSP)在骨组织生长和发育中的作用 改建。我们建议(1)建立转基因成骨细胞系 组成地表达OC和BSP的正常和定点突变 以及(2)开发体外和体内程序以评估准确的 OC和BSP在骨矿化和骨矿形成中的作用 利用这些转基因细胞进行重建。已经设计了实验 批判性地检验以下两个假设：OC提供信号 在细胞外基质(ECM)内促进破骨细胞生成 和/或骨吸收，并且该BSP在 成骨细胞和破骨细胞相互作用所需的细胞外基质 矿化阶段。高效质粒与逆转录病毒 将开发载体用于禽类OC的转导和筛选 和特定成骨细胞系中的BSP基因。禽类基因的利用 提供了独特的优势，鸟类之间的序列差异 基因和它们的小鼠对应基因提供了一种识别两者的方法 分子和抗体从禽类基因中探测内源性小鼠 产品。OC和BSP在胞外的结构/功能关系 基质组装、矿化和成骨细胞与或 对ECM的反应将被确定。体外研究将集中在 细胞外基质的形成和矿化 矿化条件，同时制备规模水平的重组 突变蛋白将被准备用于筛选它们在促进 体外培养破骨细胞的细胞黏附和表型效应 成骨细胞。平行实验将检验结构/功能 OC和BSP在细胞外基质中的表达与破骨细胞功能的关系 和破骨细胞的形成。将使用体内和体外两种方法 在这些研究中。对于体内研究，转基因细胞将是 在新的三维培养装置中生长，然后植入 变成正常的同基因小鼠。组织形态测量学参数将进行比较 为了确定特定转基因的表达对 特定ECM蛋白质和矿物质的化学计量积累 在组织内。用于重塑转基因组织的研究 体内发育将通过生化、组织形态计量学、 破骨细胞发育的原位杂交和分子分析 功能性骨吸收。

The overall goal of this proposal is to develop new experimental strategies to elucidate the functional roles of the bone matrix molecules, osteocalcin (OC) and bone sialprotein (BSP) during bone tissue growth and remodeling. We propose (1) to develop transgenic osteoblast cell lines that constitutively express normal and site-directed mutants of OC and BSP and (2) to develop in vitro and in vivo procedures to assess the precise functional roles that OC and BSP play in bone mineralization and remodeling using these transgenic cells. Experiments have been designed to critically test the following two hypotheses: that OC provides signals within the extracellular matrix (ECM) that promote osteoclastogenesis and/or bone resorption and that BSP provides positional information within the ECM that is required for osteoblastic and osteoclastic interaction with the mineralized phase. High efficiency plasmid and retroviral vectors will be developed for the transfection and selection of avian OC and BSP genes within specific osteoblastic cell lines. Use of avian genes offers the unique advantage that sequence differences between the avian genes and their murine counterparts provides a means to identify with both molecular and antibody probes the endogenous mouse from the avian gene products. Structure/function relationships of OC and BSP on extracellular matrix assembly, mineralization, and osteoblast interaction with, or response to the ECM will be determined. In vitro studies will focus on ECM formation and mineralization of the cell lines grown in culture under mineralizing conditions, while preparative scale levels of the recombinant mutant proteins will be prepared for screening of their role in promoting cell adherence and phenotypic effects on cultured osteoclasts and osteoblasts. Parallel experiments will examine the structure/function relationships of OC and BSP expression within ECM on osteoclast function and osteoclastogenesis. Both in vivo and in vitro approaches will be used in these studies. For the in vivo studies the transgenic cells will be grown in novel three-dimensional culture devices followed by implantation into normal syngeneic mice. Histomorphometric parameters will be compared to determine the effects of the expression of the specific transgenes on the stoichiometric accumulation of specific ECM proteins and mineral within the tissue. For studies on remodeling the transgenic tissues which develop in vivo will be examined by biochemical, histomorphometric, in situ hybridization, and molecular analysis of osteoclastic development and functional bone resorption.