GROWTH PLATE STUDIES IN THE CHONDRODYSTROPHIES

软骨营养不良的生长板研究

• 批准号: 2198070
• 负责人: WILLIAM A HORTON
• 金额: $ 14.33万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2198070
• 关键词: SDS polyacrylamide gel electrophoresis; autoradiography; bone development disorder; cartilage development; cartilage disorder; cartilage metabolism; cell differentiation; cell growth regulation; chondrocytes; chondrodystrophy; chondroitin; chondroitin sulfates; collagen; connective tissue disorder; connective tissue metabolism; electron microscopy; extracellular matrix; fibronectins; gel electrophoresis; high performance liquid chromatography; histochemistry /cytochemistry; hyaluronate; immunochemistry; in situ hybridization; keratan sulfate; microscopy; monoclonal antibody; normal ossification; nucleic acid probes; polymerase chain reaction; procollagen; protein biosynthesis; proteoglycan; radionuclides; radiotracer; scintillation counter; sulfur; tritium; western blottings

Cartilage is critical to skeletal development, growth and function. The cartilage collagen fibril which is comprised of types II, IX and XI collagen is responsible for the form and integrity of cartilage. Compared to collagen fibrils in other connective tissues, much less is known about cartilage collagen fibrils. Indeed, many questions remain unresolved about their organization and assembly, the function of minor fibril constituents, the role of the cell in regulating fibril formation, etc. We propose to use a combined genetic, cellular and molecular biology approach and a unique group of naturally occurring human mutants of chondrogenesis to address these and related questions. Studies over the past 5 years have identified a group of human chondrodysplasias called Dominant Cartilage Matrix Fibril Dysplasias (DCMFDs) which share dominant inheritance and qualitatively similar clinical and pathologic phenotypes. The latter includes structural abnormalities of cartilage collagen fibrils, electrophoretic abnormalities of cartilage collagens and enlargement of RER. We hypothesize that DCMFDs result from heterozygous mutations of the genes encoding the constituent chains of types II, IX and XI collagens which alter biosynthetic processing of the molecules and fibrillogenesis leading to a defective cartilage matrix. The goal of the continuation of this project is to characterize a spectrum of DCMFD mutations and examine their effects on chondrogenesis to elucidate the molecular basis of these disorders and their phenotypic variability and gain insight into normal cartilage collagen fibril biology. The analyses of each of over 40 DCMFD cases will be done in a stepwise fashion centered around a chondrocyte culture system developed by the PI which provides cartilage-like tissue, newly synthesized collagens, DNA and RNA for analysis. Analysis of collagens will identify "candidate" collagen genes for further study. Southern and Northern blot analyses of genomic DNA and mRNA will then be used to screen candidate genes for gross mutations. To detect the mire likely subtle mutations, cDNA will be synthesized from total cellular RNA. Sequences of interest will then be amplified by PCR and analyzed by chemical cleavage to detect differences (possible mutations) between the candidate and normal cDNAs. "Suspect" cDNAs will then be sequenced. Once a mutation is identified and confirmed in genomic DNA, the mutant gene product will be further characterized biochemically; and the adverse effects of the mutation on collagen biosynthetic processing and fibril structure and organization will be investigated in vitro using pulse-chase, precursor localization, immunoelectron microscopic and rotary shadowing techniques. As mutations become known and mutation:phenotype correlations become possible, short segments of genomic DNA from formalin-fixed tissues of other DCMFD patients will be amplified by PCR to confirm the correlations and better define the molecular basis of the phenotypic variability. 10-15 mutations are expected to be defined.

软骨对于骨骼发育，生长和功能至关重要。这 软骨胶原原纤维由II型，IX和XI组成 胶原蛋白负责软骨的形式和完整性。比较的 对于其他结缔组织中的胶原蛋白纤维，关于 软骨胶原纤维。确实，许多问题仍未解决 他们的组织和集会，次要原纤维成分的功能， 细胞在调节原纤维形成等中的作用。我们建议使用 一种遗传，细胞和分子生物学方法，独特的 天然发生的软骨发生的人类突变体 这些和相关的问题。 在过去的5年中，研究确定了一群人 软骨浮肿称为显性软骨基质原纤维异常增生 （DCMFD）具有主导性继承和定性相似 临床和病理表型。后者包括结构 软骨胶原纤维，电泳异常的异常 软骨胶原蛋白和RER的扩大。我们假设DCMFD 由编码成分的基因的杂合突变产生 II型，IX和XI胶原的链，会改变生物合成处理 分子和原纤维发生导致软骨有缺陷 矩阵。该项目延续的目的是表征 DCMFD突变的光谱并检查其对软骨生成的影响 阐明这些疾病的分子基础及其表型 变异性并深入了解正常软骨胶原纤维生物学。 将在40多个DCMFD案例中的分析进行逐步进行 时尚围绕PI开发的软骨细胞培养系统 提供软骨状组织，新合成的胶原蛋白，DNA和 RNA进行分析。胶原蛋白的分析将识别“候选”胶原蛋白 用于进一步研究的基因。基因组DNA的南部和北印迹分析 然后，mRNA将用于筛选候选基因进行总突变。 为了检测泥浆可能微妙的突变，将从 总细胞RNA。然后，感兴趣的序列将通过PCR和 通过化学裂解分析以检测差异（可能的突变） 在候选和正常cDNA之间。 “可疑” cdnas将是 测序。一旦在基因组DNA中鉴定并确认突变， 突变基因产物将进一步以生化为特征。和 突变对胶原蛋白生物合成过程的不利影响和 原纤维结构和组织将在体外研究 脉冲追踪，前体定位，免疫电子显微镜和旋转 阴影技术。随着突变的已知和突变：表型 相关性变得可能是可能的，基因组DNA的短段 其他DCMFD患者的福尔马林固定组织将通过PCR扩增至 确认相关性并更好地定义分子基础 表型变异性。预计将定义10-15个突变。