BASIC FIBROBLAST GROWTH FACTOR EXPRESSION IN BONE CELLS

碱性成纤维细胞生长因子 (FGF-2) 是成骨细胞和骨髓基质细胞的有效有丝分裂原，由骨细胞合成。 FGF-2缺失小鼠的骨小梁体积显着减少，骨髓培养物中骨结节形成减少，并且对各种激动剂的反应减少破骨细胞形成。我们假设 FGF-2 调节成骨细胞的复制和分化，FGF-2 基因的破坏会导致骨形成受损。 此外，由于支持细胞（成骨细胞）不足或破骨细胞生成重要因子的产生减少，破骨细胞形成减少。 目的 1：阐明内源性 FGF-2 在骨形成中的作用。 我们将比较没有 FGF-2 基因的小鼠 (FGF-2-/-) 与野生型 (FGF-2 / ) 小鼠。 对不同年龄的小鼠进行动态组织形态测定和骨密度测定，以确定FGF-2(-/-)小鼠骨小梁体积减少的发生率。 我们将检查来自 FGF-2 (-/-) 和 FGF-2 (/) 的骨髓基质细胞培养物和颅骨细胞培养物中的成骨细胞分化。 我们将确定 FGF-2 缺失对早期和成熟成骨细胞分化的基因标志物的影响，包括 CBFA1、1 型胶原、碱性磷酸酶和骨钙素。 我们将检查支持成骨细胞分化的基因的表达，这些基因可能位于 FGF-2 的下游，包括 BMP2 和 TGFbeta。 我们将通过测量 DNA、胸苷标记以及胶原蛋白和非胶原蛋白中脯氨酸的掺入情况来比较颅骨的骨形成。 我们将确定体内施用 FGF-2 是否可以逆转 FGF-2 (-/-) 小鼠的骨质流失。 目标 2：确定 FGF-2 缺失小鼠的破骨细胞形成和骨吸收是否发生改变。 我们将比较激动剂使用骨髓诱导破骨细胞形成的能力，以及来自 FGF-2 (-/-) 和 FGF-2 (/) 小鼠的原代颅骨富集的成骨细胞和脾细胞的共培养模型。 我们将通过 CFU-GM 测定来测量这些动物中的破骨细胞前体数量。 我们将检查对破骨细胞形成至关重要的基因的表达，包括骨保护素配体 (OPGL)、骨保护素 (OPG)、NFKB 受体激活剂 (RANK) 和巨噬细胞集落刺激因子。 我们将检查对骨转换很重要的基因的表达，这些基因可能位于 FGF-2 的下游，包括 PGHS-2、IGF-I、胶原酶。 为了评估骨吸收，我们将测量未刺激和刺激状态下预标记颅骨的 45Ca 释放。 对 FGF-2 缺失小鼠的骨形成和吸收的分析不仅有助于了解这种生长因子在骨重塑中的生理和病理作用，而且有助于开发 FGF-2 在骨质疏松症等骨疾病中的治疗用途。

Basic fibroblast growth factor (FGF-2), a potent mitogen for osteoblasts and marrow stromal cells, is synthesized by bone cells. FGF-2 null mice have marked decreased trabecular bone volume, decreased bone nodule formation in marrow cultures and reduce osteoclast formation in response to a variety of agonists. We hypothesize that FGF-2 regulates osteoblast replication and differentiation and that disruption of the FGF-2 gene results in impaired bone formation. In addition, osteoclast formation is reduced due to either insufficient support cells (osteoblasts), or reduced production of factors important for osteoclastogenesis. Aim 1: To elucidate the role of endogenous FGF-2 in bone formation. We will compare mice with no FGF-2 gene (FGF-2-/-) with wild type (FGF-2+/+) mice. Dynamic histomorphometry and bone densitometry will be performed on mice of different ages to determine the rate that decreased trabecular bone volume in FGF-2(-/-) mice develops. We will examine osteoblast differentiation in bone marrow stromal cell cultures and calvarial cell cultures from FGF-2 (-/-) and FGF-2 (+/+). We will determine the effect of loss of FGF-2 on gene markers of differentiation of early and mature osteoblasts including, CBFA1, type 1 collagen, alkaline phosphatase and osteocalcin. We will examine the expression of genes which support osteoblast differentiation which may be downstream of FGF-2 including BMP2 and TGFbeta. We will compare bone formation in calvariae by measuring DNA, thymidine labeling and proline incorporation into collagen and noncollagen proteins. We will determine whether in vivo administration of FGF-2 reverses bone loss in FGF-2 (-/-) mice. Aim 2: To determine whether osteoclast formation and bone resorption are altered in FGF-2 null mice. We will compare the ability of agonists to induce osteoclast formation using bone marrow, as well as coculture models of primary calvarial enriched osteoblasts and spleen cells from FGF-2 (-/-) and FGF-2 (+/+) mice. We will measure osteoclast precursor numbers in these animals by the CFU-GM assay. We will examine the expression of genes that are critical for osteoclast formation including osteoprotegerin ligand (OPGL), osteoprotegerin (OPG), receptor activator of NFKB (RANK) and macrophage colony stimulating factor. We will examine the expression of genes which are important in bone turnover which may be downstream of FGF-2 including PGHS-2, IGF-I, collagenase. To assess bone resorption, we will measure 45Ca release from prelabeled calvariae in the unstimulated and stimulated state. Analysis of bone formation and resorption in FGF-2 null mice is relevant not only to understanding the physiologic and pathologic role of this growth factor in bone remodeling, but also to facilitate the development of therapeutic usage of FGF-2 in bone disorders such as osteoporosis.