Regulation of Cranial Suture Development by TGF-B and FG

描述（由申请人提供）：颅缝闭锁是人类重要的健康问题，但其发病机制仍未得到解决。临床和实验研究表明转化生长因子- 1 （tgf - β 1）和成纤维细胞生长因子-2参与颅骨缝合融合。有证据表明，一种或两种分子对缝线融合的影响可能被noggin（一种骨形态发生蛋白拮抗剂）进一步改变。在许多人颅缝闭锁综合征中，FGF受体功能突变的增加已被报道，其中大多数综合征涉及FGF受体2 （FGFR2）。然而，tgf - β 1和FGF-2在颅骨缝合发育中的确切作用尚未得到解释。我们假设：1)在小鼠后额叶（PF）缝合发育期间，tgf - β 1和FGF-2的生物活性改变调节上覆缝合颅骨的融合；2)这些分子在小鼠后额叶（PF）缝合中的表达与先天性颅缝紧闭儿童融合缝合线中的表达相似。FGFR2和noggin的改变也可能有助于改变tgf - β 1和FGF-2的生物活性，并进一步调节缝合融合。具体目的是：1)在体内和体外模型中获取的小鼠颅骨组织中，定量tgf - β 1、FGF-2、FGFR2和noggin的基因表达。另外，用ELISA法测定体外培养基中TGF- β 1、FDF-2和noggin的蛋白水平。2)定量分析这些生长因子和受体的基因表达，这些生长因子和受体来自人类外阴颅缝闭闭患者。作为颅缝闭合标准治疗的一部分，我们将从接受开颅手术的儿童身上采集融合缝合和未闭合缝合的组织。这些数据将用于帮助确定小鼠和人类颅骨缝合系统之间的关系。3)通过调节目的1中描述的生长因子和受体的生物利用度，在体外调节小鼠颅骨缝合融合模型。我们将每个分子的重组形式或抗体添加到这些分子中，以诱导正常通畅的矢状缝线融合或阻止PF缝线的正常融合。4)探讨质粒DNA体外转染小鼠脑组织的可能性。如果成功，靶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骨缝合的发育。5)获得足够的分子生物学知识，发展成为一名独立的临床科学家。这一建议将为颅缝发育的调控提供机制见解，并有助于将小鼠和人类颅缝闭合模型联系起来。

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.