Altered Matrix-Cells and Intermolecular Interactions

描述(申请人提供)：我们在这里提出的研究是我们目前关于纤维胶原突变对细胞外基质结构和细胞行为的影响的研究的继续，特别强调II型胶原的突变及其对软骨的影响。这项研究的长期目标是利用三维软骨样结构来确定突变的胶原分子对细胞外基质结构和细胞行为的影响，并确定细胞和基因治疗的目标参数，以改变在胶原突变存在时出现的异常表型。我们的假设是，纤维性胶原蛋白的突变不仅会改变细胞外基质的结构，还会影响细胞的行为，不同的胶原蛋白突变体对治疗方法的成功要求不同。为了满足我们的长期目标和验证所述的假设，我们制定了以下具体目标：(1)建立一个具有生物学意义的实验模型，研究胶原突变对病变组织退化和修复的影响；(2)确定胶原突变表达抑制对异常结缔组织重塑的影响；(3)确定细胞治疗所需的条件，以覆盖胶原突变引起的病理变化。胶原基因突变的显性负面影响的治疗进展的一个根本障碍是缺乏关于需要达到的最低条件的信息，以推动受影响的组织对细胞或基因治疗做出反应，使其重塑为正常结构。在我们的研究中，我们将通过创建一个生物相关的模型来解决这个问题，该模型将类似于软骨的复杂性。这个模型将由工程细胞组成，除了内源性野生型II型前胶原外，这些细胞还将有条件地表达编码在各种形式的软骨发育不良患者中发现的II型前胶原突变的cDNAs结构。这些细胞将被用来在细胞培养条件下和裸鼠体内产生软骨样结构。随后，在切断突变的cDNA或模拟表达野生型II型前胶原的细胞的实验后，将研究在存在突变的II型胶原变体的情况下形成的软骨样结构的形态、生物学和生物力学特性的变化。通过使用基因和细胞治疗的模型，我们提出的研究将确定软骨组织修复和再生的内在能力。此外，这些研究将为设计治疗方法提供重要信息，不仅抵消II型胶原突变的主要负面影响，而且最有可能抵消与骨骼组织遗传性疾病相关的其他胶原性和非胶原性细胞外基质大分子突变的影响。因此，拟议的研究与公共卫生具有很高的相关性。

DESCRIPTION (provided by applicant): Research we propose here is a continuation of our current study on the effects of mutations in fibrillar collagens on the structure of extracellular matrices and behavior of cells with special emphasis on mutations in collagen II and their effects on cartilage. The broad, long-term objective of the proposed study is to employ three-dimensional cartilage-like constructs to determine effects of the presence of mutant collagen molecules on the structure of ECM, cell behavior, and to define target parameters for cell and gene therapies that should be reached to change abnormal phenotypes developed in the presence of collagen mutants. Our hypotheses are that mutations in fibrillar collagens alter not only the structure of extracellular matrices, but also impact the behavior of cells, and that requirements for therapy approaches to be successful vary for different collagen mutants. To meet our long-term objectives and test stated hypotheses we formulated the following Specific Aims: (1) To create a biologically relevant experimental model to study effects of collagen mutants on degeneration and repair of affected tissues, (2) To determine effects of suppression of expression of collagen mutants on remodeling of abnormal connective tissue, (3) To define conditions for cell therapies needed to override pathological changes caused by collagen mutants. A fundamental barrier to move forward development of therapies for dominant negative effects of mutations in collagen genes is a lack of information about minimal conditions needed to be achieved to drive affected tissues toward their remodeling into normal structures in response to cell or gene therapies. In our studies we will address this problem by creating a biologically relevant model, which will resemble the complexity of cartilage. This model will consist of engineered cells that, in addition to endogenous wild type procollagen II will conditionally express cDNA constructs encoding procollagen II mutants found in patients with various forms of chondrodysplasias. These cells will be employed to create cartilage-like constructs in cell culture conditions and in athymic nude mice. Subsequently, changes in morphological, biological, and biomechanical characteristics of the cartilage-like constructs formed in the presence of mutant collagen II variants will be studied after switching off mutant cDNAs or after experiments simulating "delivery" of cells expressing wild type procollagen II. By employing models for gene and cell therapies, studies we propose will determine intrinsic capacity of cartilaginous tissues to repair and regenerate. Moreover, these studies will provide important information for designing therapeutic approaches to counterbalance not only dominant negative effects of mutations in collagen II but also, most likely, effects of mutations in other collagenous and noncollagenous extracellular matrix macromolecules that are associated with heritable diseases of skeletal tissues. Thus, the relevance of the proposed study to public health is high.