BASIC FIBROBLAST GROWTH FACTOR EXPRESSION IN BONE CELLS

碱性成纤维细胞生长因子（FGF-2）是骨细胞合成的一种有效的成骨细胞和骨髓基质细胞的有丝分裂原。 FGF-2基因敲除小鼠对多种激动剂的反应是骨小梁体积显著减少，骨髓培养物中骨结节形成减少，破骨细胞形成减少。我们假设FGF-2调节成骨细胞的复制和分化，FGF-2基因的破坏导致骨形成受损。 此外，破骨细胞形成减少是由于支持细胞（成骨细胞）不足，或破骨细胞生成重要因子的产生减少。 目的1：阐明内源性FGF-2在骨形成中的作用。 我们将比较无FGF-2基因的小鼠（FGF-2-/-）与野生型（FGF-2+/+）小鼠。 将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（-/-）小鼠中骨小梁体积减少的发生率。 我们将研究成骨细胞分化的骨髓基质细胞培养和颅骨细胞培养的FGF-2（-/-）和FGF-2（+/+）。 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CBFA 1、1型胶原、碱性磷酸酶和骨钙素。 我们将检测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BMP 2和TGF β。 我们将通过测量DNA、胸苷标记和脯氨酸掺入胶原和非胶原蛋白来比较颅骨的骨形成。 我们将确定体内施用FGF-2是否逆转FGF-2（-/-）小鼠的骨丢失。 目的2：探讨FGF-2基因敲除小鼠破骨细胞形成和骨吸收的变化。 我们将使用骨髓以及原代颅骨富集成骨细胞和FGF-2（-/-）和FGF-2（+/+）小鼠脾细胞的共培养模型，比较激动剂诱导破骨细胞形成的能力。 我们将通过CFU-GM测定来测量这些动物中破骨细胞前体的数量。 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）、NF κ B受体激活剂（RANK）和巨噬细胞集落刺激因子。 我们将检测在骨转换中重要的基因的表达，这些基因可能是FGF-2的下游，包括PGHS-2、IGF-I、胶原酶。 为了评估骨吸收，我们将在未刺激和刺激状态下测量预标记的颅骨中的45 Ca释放。 分析FGF-2基因敲除小鼠的骨形成和再吸收不仅与理解这种生长因子在骨重建中的生理和病理作用有关，而且还有助于开发FGF-2在骨疾病如骨质疏松症中的治疗用途。

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.