BASIC FIBROBLAST GROWTH FACTOR EXPRESSION IN BONE CELLS

碱性成纤维细胞生长因子-2是成骨细胞和骨髓基质细胞的强有力的有丝分裂原，是由骨细胞合成的。成纤维细胞生长因子-2基因缺失小鼠的骨小梁体积显著减少，骨髓培养中骨结节的形成减少，破骨细胞的形成对各种激动剂的反应减少。我们假设成纤维细胞生长因子-2调控成骨细胞的复制和分化，而成纤维细胞生长因子-2基因的破坏会导致骨形成障碍。此外，破骨细胞的形成减少是由于支持细胞(成骨细胞)不足，或者是破骨细胞生成的重要因子产生减少。目的1：探讨内源性成纤维细胞生长因子-2在骨形成中的作用。我们将比较不携带成纤维细胞生长因子-2基因的小鼠(成纤维细胞生长因子2-/-)和野生型(成纤维细胞生长因子-2+/+)小鼠。将对不同年龄的小鼠进行动态组织形态计量学和骨密度测量，以确定成纤维细胞生长因子-2(-/-)小鼠的骨小梁体积减少的速度。我们将从成纤维细胞生长因子-2(-/-)和成纤维细胞生长因子-2(+/+)中检测成骨细胞在骨髓基质细胞培养和颅骨细胞培养中的分化。我们将测定成纤维细胞生长因子-2缺失对早期成骨细胞和成熟成骨细胞分化相关基因标志物CBFA1、I型胶原、碱性磷酸酶和骨钙素的影响。我们将研究支持成骨细胞分化的基因的表达，这些基因可能位于成纤维细胞生长因子-2的下游，包括骨形成蛋白2和转化生长因子β。我们将通过测量DNA、胸腺嘧啶核苷标记以及胶原和非胶原蛋白中的脯氨酸掺入来比较头盖骨的骨形成。我们将确定体内应用成纤维细胞生长因子-2是否能逆转成纤维细胞生长因子-2(-/-)小鼠的骨丢失。目的：检测成纤维细胞生长因子-2基因缺失小鼠破骨细胞的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，以及来自成纤维细胞生长因子-2(-/-)和成纤维细胞生长因子-2(+/+)小鼠的原代富含颅骨的成骨细胞和脾细胞的共培养模型。我们将通过CFU-GM检测这些动物的破骨细胞前体数量。我们将检测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。我们将研究可能在成纤维细胞生长因子-2下游的骨转换中重要的基因的表达，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先标记的头盖骨的45Ca释放。分析成纤维细胞生长因子-2基因缺失小鼠的骨形成和骨吸收，不仅有助于了解该生长因子在骨重建中的生理和病理作用，而且有助于促进成纤维细胞生长因子-2在骨质疏松等骨病治疗中的应用。

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.