Altered Matrix-Cells and Intermolecular Interactions

描述（由申请人提供）：我们在这里提出的研究是我们目前关于纤维性胶原突变对细胞外基质结构和细胞行为影响的研究的延续，特别强调胶原II突变及其对软骨的影响。该研究的广泛而长期的目标是采用三维软骨样结构来确定突变胶原分子对ECM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，以改变在胶原突变体存在下产生的异常表型。我们的假设是，纤维性胶原的突变不仅改变细胞外基质的结构，而且影响细胞的行为，并且不同的胶原突变对治疗方法的要求是不同的。为了实现我们的长期目标和验证既定假设，我们制定了以下具体目标：(1)创建一个生物学相关的实验模型，研究胶原突变体对受影响组织的变性和修复的影响；(2)确定抑制胶原突变体表达对异常结缔组织重塑的影响；(3)确定克服胶原突变体引起的病理变化所需的细胞治疗条件。胶原蛋白基因突变的主要负面影响的治疗方法发展的一个基本障碍是缺乏关于驱动受影响组织在细胞或基因治疗下重塑为正常结构所需的最小条件的信息。在我们的研究中，我们将通过创建一个生物学上相关的模型来解决这个问题，这个模型将类似于软骨的复杂性。该模型将由工程细胞组成，这些细胞除了内源性野生型前胶原II外，还会有条件地表达编码各种形式软骨发育不良患者中发现的前胶原II突变体的cDNA构建物。这些细胞将用于在细胞培养条件下和胸腺裸鼠中制造软骨样结构。随后，在关闭突变型cdna或模拟表达野生型前胶原II细胞的“递送”实验后，将研究在突变型II型胶原变体存在下形成的软骨样结构的形态学、生物学和生物力学特征的变化。通过采用基因和细胞治疗模型，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，不仅可以抵消II型胶原突变的主要负面影响，而且很可能还可以抵消与骨组织遗传性疾病相关的其他胶原和非胶原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的相关性很高。

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.