Regulation of Cranial Suture Development by TGF-B and FG

描述(申请人提供)：颅缝早闭是人类的一个重要的健康问题，但其发病机制和机制仍未解决。临床和实验研究表明转化生长因子-β1和成纤维细胞生长因子-2在颅缝融合中起重要作用。有证据表明，一个或两个分子对缝合融合的影响可能会被骨形态发生蛋白拮抗剂noggin进一步改变。在许多人类颅缝融合综合征中已经报道了成纤维细胞生长因子受体功能突变的获得，这些综合征中的大多数与成纤维细胞生长因子受体2(FGFR2)有关。然而，转化生长因子-β1和成纤维细胞生长因子-2在颅缝发育中的确切作用尚不清楚。我们假设：1)在小鼠额后缝发育期间，转化生长因子-β1和成纤维细胞生长因子-2的生物活性改变调节了上覆缝颅骨的融合；2)这些分子在小鼠融合的额后缝中的表达与先天性颅缝早闭儿童融合缝中的表达相似。FGFR2和noggin的改变也可能有助于改变转化生长因子-β1和成纤维细胞生长因子-2的生物学活性，从而进一步调节缝线融合。目的：1)定量检测体内和体外模型小鼠颅骨组织中转化生长因子-β1、成纤维细胞生长因子-2、成纤维细胞生长因子-2和noggin的基因表达。此外，体外培养上清液中的转化生长因子-β1、fdf-2和noggin的蛋白水平将用酶联免疫吸附试验测定。2)定量分析同种生长因子及其受体在人先天性颅缝早闭患者中的基因表达。我们将从接受开颅手术的儿童的融合缝合线和专利缝合线中获取组织，作为颅缝融合症标准治疗的一部分。这些数据将被用来帮助确定小鼠和人类颅缝系统之间的关系。3)通过调节目标1中所述的生长因子和受体的生物利用度，在体外调节小鼠颅缝融合。我们将每个分子或抗体的重组形式添加到这些分子上，以诱导正常开放的矢状缝融合或阻断PF缝的正常融合。4)探讨小鼠颅骨组织体外转染质粒DNA的可能性。如果成功，靶向生长因子和受体的基因操作可能在未来的研究中用于调节颅缝的发育。5)掌握足够的分子生物学知识，发展成为独立的临床医生-科学家。这一建议将为颅缝发育的调节提供机械性的见解，并有助于将颅缝融合的小鼠和人类模型联系起来。

DESCRIPTION (provided by applicant): Craniosynostosis is a significant health issue in humans, yet the pathogenesis and the mechanism of craniosynostosis are still unsolved. Clinical and experimental studies have implicated Transforming Growth Factor-a 1 (TGF-beta1) and Fibroblast Growth Factor-2 in cranial suture fusion. There is evidence that the effect of one or both molecules on suture fusion may be further altered by noggin, a bone morphogenetic protein antagonist. Gain of function mutations in FGF receptors have been reported in numerous human craniosynostosis syndromes, with the majority of these syndromes implicating FGF receptors 2 (FGFR2). However, the precise role of TGF-beta1 and FGF-2 in cranial suture development has not been explained. We hypothesize that 1) during the period of posterior frontal (PF) suture development in the mouse, altered biologic activity of TGF-beta1 and FGF-2 regulates fusion of the overlying suture calvaria, and 2) expression of these molecules in the fused PF suture in mice parallels that in fused sutures from human children with congenital craniosynostosis. Alteration in FGFR2 and noggin may also contribute to alter biologic activity of TGF-Beta1 and FGF-2 and further regulate suture fusion. Specific aims are 1) To quantity gene expression of TGF-beta1, FGF-2, FGFR2, and noggin in mouse cranial tissues harvested from both in vivo and in vitro models. Additionally, protein levels of TGF- beta1, FDF-2, and noggin will be measured in culture media in vitro using ELISA. 2) To quantitate gene expression of these same growth factors and receptors harvested from human patients with cogenital craniosynostosis. We will harvest tissue from both a fused suture and a patent suture from children undergoing craniotomy as part of the standard treatment for craniosynotosis. These data will be used to help determine the relationship between the mouse and human cranial suture systems. 3) To modulate cranial suture fusion in the mouse model, in vitro by modulating the bioavailability of the growth factor and receptors described in Aim 1. We will add recombinant forms of each molecule or antibodies to these molecules to either induce fusion of the normally patent sagittal suture or block the normal fusion of the PF suture. 4) To investigate the possibility of in vitro plasmid DNA transfection of mouse cranial tissue. If successful, gene manipulation of the target growth factors and receptors may be used to regulate cranial suture development in future studies. 5) To gain adequate knowledge of molecular biology to develop into an independent clinician-scientist. This proposal will provide mechanistic insights into the regulation of cranial suture development, and help to relate mouse and human models of craniosynostosis.