Regulation of Cranial Suture Development by TGF-Beta and Fibroblast Growth Factor

TGF-β 和成纤维细胞生长因子对颅缝发育的调节

• 批准号: 7224143
• 负责人: ARUN K GOSAIN
• 金额: $ 13.19万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-10 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7224143
• 关键词: 3 year old; Antibodies; Basic Science; Biological Availability; Biology; Birth; Blocking Antibodies; Bone Morphogenetic Proteins; Calvaria; Cephalic; Child; Clinical; Craniosynostosis; Craniotomy; Culture Media; Data; Development; Dura Mater; Educational Curriculum; Enzyme-Linked Immunosorbent Assay; Evaluation; Fibroblast Growth Factor; Fibroblast Growth Factor 2; Fibroblast Growth Factor Receptor 2; Fibroblast Growth Factor Receptors; Future; Gene Expression; Genes; Green Fluorescent Proteins; Growth Factor Receptors; Harvest; Health; Human; Immune Sera; In Vitro; Investigation; Joint structure of suture of skull; Knowledge; Laboratories; Legal patent; Length; Lipofectamine; Maintenance; Mammalian Cell; Measures; Mediating; Mentorship; Mesenchyme; Messenger RNA; Modeling; Molecular Biology; Molecular Genetics; Mus; Organ Culture Techniques; Pathogenesis; Patients; Plasmid Cloning Vector; Plasmids; Procedures; Proteins; Recombinants; Regulation; Reporter Genes; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Role; Rotation; Scientist; Specimen; Standards of Weights and Measures; Surgical sutures; Syndrome; System; Time; Tissue Harvesting; Tissues; Transfection; Transforming Growth Factor beta; Transforming Growth Factors; brain tissue; day; design; gain of function mutation; human TGFB1 protein; in vitro Model; in vivo; in vivo Model; insight; knowledge base; mouse model; noggin protein; plasmid DNA; programs; receptor; research study; skills; suture fusion

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.

描述（申请人提供）： 颅缝早闭是人类的一个重要健康问题，但颅缝早闭的发病机制和机制仍未得到解决。临床和实验研究表明转化生长因子-a 1 (TGF-β1) 和成纤维细胞生长因子-2 与颅缝融合有关。有证据表明，一种或两种分子对缝线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。据报道，许多人类颅缝早闭综合征中存在 FGF 受体功能突变，其中大多数综合征与 FGF 受体 2 (FGFR2) 有关。然而，TGF-β1和FGF-2在颅缝发育中的确切作用尚未得到解释。我们假设：1) 在小鼠额后 (PF) 缝线发育期间，TGF-β1 和 FGF-2 生物活性的改变调节了覆盖颅骨缝线的融合，2) 这些分子在小鼠 PF 融合缝线中的表达与患有先天性颅缝早闭的人类儿童的融合缝线中的表达相似。 FGFR2 和 noggin 的改变也可能有助于改变 TGF-Beta1 和 FGF-2 的生物活性，并进一步调节缝线融合。具体目标是 1) 定量从体内和体外模型收获的小鼠颅组织中 TGF-β1、FGF-2、FGFR2 和 noggin 的基因表达。此外，将使用 ELISA 测量体外培养基中 TGF-β1、FDF-2 和 noggin 的蛋白质水平。 2) 定量从患有先天性颅缝早闭的人类患者中采集的这些相同生长因子和受体的基因表达。作为颅缝早闭标准治疗的一部分，我们将从接受开颅手术的儿童身上采集融合缝合线和专利缝合线的组织。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) 通过调节目标 1 中描述的生长因子和受体的生物利用度，在体外调节小鼠模型中的颅缝融合。我们将向这些分子添加每个分子或抗体的重组形式，以诱导正常专利矢状缝的融合或阻断 PF 缝的正常融合。 4)探讨质粒DNA体外转染小鼠颅组织的可能性。如果成功，目标生长因子和受体的基因操作可用于在未来的研究中调节颅缝发育。 5) 获得足够的分子生物学知识，发展成为一名独立的临床医生科学家。该提案将为颅缝发育的调节提供机制见解，并有助于将小鼠和人类的颅缝早闭模型联系起来。

项目成果

TGF-beta1 RNA interference in mouse primary dura cell culture: downstream effects on TGF receptors, FGF-2, and FGF-R1 mRNA levels.

小鼠原代硬脑膜细胞培养中的 TGF-β1 RNA 干扰：对 TGF-β1 受体、FGF-2 和 FGF-R1 mRNA 水平的下游影响。

• DOI: 10.1097/prs.0b013e3181b98947
• 发表时间: 2009
• 期刊: Plastic and reconstructive surgery
• 影响因子: 3.6
• 作者: Gosain,ArunK;Machol4th,JacquesA;Gliniak,Christy;Halligan,NadineLN
• 通讯作者: Halligan,NadineLN

Detection of apoptosis in fusing versus nonfusing mouse cranial sutures.

融合与非融合小鼠颅缝中细胞凋亡的检测。

• DOI: 10.1097/01.scs.0000168759.61947.7f
• 发表时间: 2005
• 期刊: The Journal of craniofacial surgery
• 作者: Agresti,Michael;Gosain,ArunK
• 通讯作者: Gosain,ArunK

Real-time reverse transcriptase polymerase chain reaction: an improvement in detecting mRNA levels in mouse cranial tissue.

实时逆转录酶聚合酶链式反应：检测小鼠颅组织中 mRNA 水平的改进​​。

• DOI: 10.1097/01.prs.0000219339.82624.b9
• 发表时间: 2006
• 期刊: Plastic and reconstructive surgery
• 影响因子: 3.6
• 作者: Singh,Rashmi;Recinos,RenéF;Agresti,Michael;Schaefer,RichardB;Bosbous,Mark;Gosain,ArunK
• 通讯作者: Gosain,ArunK

Nonviral transfection of mouse calvarial organ in vitro using Accell-modified siRNA.

使用 Accell 修饰的 siRNA 对小鼠颅骨器官进行体外非病毒转染。

• DOI: 10.1097/prs.0b013e3181c82df1
• 发表时间: 2010
• 期刊: Plastic and reconstructive surgery
• 影响因子: 3.6
• 作者: Gupta,AshimK;Eshraghi,Yashar;Gliniak,Christy;Gosain,ArunK
• 通讯作者: Gosain,ArunK