Genetic and Molecular Insights Into Cartilage Regeneration, Primary and Posttraum

对原发性和创伤后软骨再生的遗传和分子见解

• 批准号: 8840890
• 负责人: MUHAMMAD FAROOQ RAI
• 金额: $ 8.77万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8840890
• 关键词: Address; Affect; Age; Age-Months; Aging; Anatomy; Animals; Arthritis; Basic Science; Biological Assay; Biology; Bone Growth; Candidate Disease Gene; Cartilage; Cartilage injury; Cell Culture Techniques; Cell Cycle; Cell physiology; Cellular biology; Chondrocytes; Chromosome Mapping; Chromosomes; Collaborations; Collection; Complex; DNA; DNA Repair; Degenerative polyarthritis; Development; Diabetes Mellitus; Disease; Ear; Ear Cartilages; Epiphysial cartilage; Gene Expression; Genes; Genetic; Genetic Variation; Genome; Genomics; Genotype; Healed; Health; Heritability; Histology; Human; Human Genome; Inbred Mouse; Inbred Strain; Inbred Strains Mice; Inbreeding; Individual; Injury; Joints; Knee; Laboratories; MRL/MpJ Mouse; Maps; Medial meniscus structure; Mentors; Mentorship; Mesenchymal Stem Cells; Messenger RNA; Methodology; Methods; MicroRNAs; Modeling; Molecular; Molecular Biology; Molecular Genetics; Monitor; Mus; Natural regeneration; Neurobiology; Obesity; Operative Surgical Procedures; Orthopedic Surgery procedures; Outcome; Pathway interactions; Patient risk; Phase; Phenotype; Population; Population Genetics; Predisposition; Property; RNA; Recombinant Inbred Strain; Recombinants; Recruitment Activity; Research Design; Research Personnel; Resistance; Rhubarb food; Role; Severities; Signal Transduction; Single Nucleotide Polymorphism; Slide; Stem cells; Stratification; Techniques; Technology; Testing; Therapeutic Intervention; Thick; Time; Tissues; Traumatic Arthropathy; Variant; Wound Healing; age group; age related; articular cartilage; base; biomechanical engineering; bone; cartilage degradation; cartilage development; cartilage regeneration; cartilage repair; follow-up; genetic approach; genetic resource; genetic strain; genetic variant; healing; insight; juvenile animal; laser capture microdissection; long bone; microCT; mouse model; novel; regenerative; repaired; response; sex; tissue regeneration; tissue repair; trait; wound

DESCRIPTION (provided by applicant): Genetic and Molecular Insights into Cartilage Regeneration, Primary and Posttraumatic Osteoarthritis Background and Rationale: This proposal is from Dr. M. Farooq Rai, a basic science investigator in the field of cartilage biology and osteoarthritis (OA). The mentored-phase will be completed under the mentorship of i) Dr. Linda J. Sandell, a cartilage biologist in the Departments of Orthopaedic Surgery and Cell Biology, ii) Dr. James M. Cheverud, a population geneticist and morphologist in the Department of Anatomy and Neurobiology, iii) Dr. Mathew J. Silva, a biomechanical engineer and bone biologist in the Department of Orthopaedic Surgery and iv) Dr. Ingrid Borecki, a biostatistician at the Division of Statistical Genomics. Using inbred strains related to the "super healing" mouse, we have defined phenotypes of ear wound healing and articular cartilage regeneration and have established a strong positive correlation between these two phenotypes. Furthermore, proof-of-concept studies strongly suggest that the ability to heal articular cartilage is positively correlaed with protection from posttraumatic OA. This unique genetic resource (i.e. recombinant inbred lines) will be used to systematically identify genes that contribute to cartilage regeneration and thus to age-related primary OA and posttraumatic OA. The methods used to undertake these specific aims rely on the availability of recombinant inbred strains of LG/J and SM/J with different abilities to regenerate cartilage and ear tissue combined with mouse surgical techniques for articular cartilage injury (full thickness) and a model of posttraumatic OA, destabilization of the medial meniscus (DMM - an OA model). Functional analyses of chondrocytes and stem cells will be performed in the strains that are extremes of healing. The outcome of this project will be the identification of genes affecting OA development and articular cartilage regeneration in mice, providing high-quality candidates for probing the human genome. These recombinant inbred lines were established by one of my mentors Dr. James Cheverud and have been extensively used to study other complex diseases such as obesity and diabetes in addition to tissue regeneration and long bone growth. Over the last 3.5 years, in collaboration, we have established the phenotype of some of these strains for cartilage regeneration and OA. Hypothesis: Gene variants can be identified in recombinant inbred mouse strains that will ultimately correlate with susceptibility to and protection from age-related primary OA and posttraumatic OA. We have developed the hypothesis that the ability to regenerate articular cartilage is positively genetically correlated with the ability to regenerate ear tissues (Rai et a., Arthritis Rheum 64:2300-10. 2012). We also began a study of the relationship between the ability to regenerate articular cartilage and susceptibility to OA, finding an inverse correlation between articular cartilage regeneration and OA (Hashimoto et al., Osteoarthritis Cartilage 20:562-71. 2012). We have also analyzed expression of candidate genes through branched-chain DNA technology on tissue lysates obtained from histological sections of eight strains. The expression of several genes was significantly heritable among strains. Four genes representing DNA repair (Xrcc2, Pcna) and Wnt signaling (Axin2, Wnt16) pathways were significantly positively correlated with both phenotypes suggesting a common genetic basis of tissue healing (under review in G3). The guiding hypothesis for this proposal is that genes can be identified that are involved in cartilage repair that will also be involved in posttraumatic OA. We propose to follow up these exciting results by gene mapping, analysis of cell function, and identification of specific genes involved in cartilage repair and OA. Study Design: The study contains four specific aims: (1) Determine phenotypic differences in young and old LGXSM recombinant inbred lines for articular cartilage regeneration and development of primary OA and posttraumatic OA. (2) Map genetic variation in young and old LGXSM recombinant inbred lines for articular cartilage regeneration and development of primary OA and posttraumatic OA, mapping QTLs for all traits to 10 cM genomic intervals. (3) Delineate molecular differences in LGXSM recombinant inbred lines for articular cartilage regeneration and development of primary OA and posttraumatic OA. (4) Monitor intrinsic functional differences in chondrocytes and mesenchymal stem cells in selected LGXSM recombinant inbred lines. Significance of Outcomes: We will be able to identify genes or groups of genes that govern tissue regeneration (ear wound and knee cartilage and protection from getting age-related primary and posttraumatic OA. Genes that playa a role in OA are unknown and studies in human address only primary OA, even then, have yielded little information, primarily because there will be many genes with small effects. This limitation can be addressed with our genetic mouse models. Finally, the cellular and molecular differences between healer and non-healer strains will provide novel insights into the mechanisms of regeneration and degermation of cartilage. Applied to human, these findings could facilitate better stratification of patient risk for progression of OA and identify possible targets for therapeutic intervention.

描述(由申请人提供)：对软骨再生、原发和创伤后骨关节炎的遗传学和分子洞察背景和原理：这项建议由软骨生物学和骨关节炎(OA)领域的基础科学研究员M.Farooq Rai博士提出。指导阶段将在以下导师的指导下完成：i)矫形外科和细胞生物系的软骨生物学家Linda J.Sandell博士，ii)解剖和神经生物学系的群体遗传学家和形态学家James M.Cheverud博士，iii)矫形外科的生物力学工程师和骨生物学家Mathew J.Silva博士，以及iv)Ingrid Borecki博士，生物统计学家 统计基因组学部。利用与“超级愈合”小鼠相关的近交系小鼠，我们定义了耳部伤口愈合和关节软骨再生的表型，并建立了这两种表型之间的强正相关关系。此外，概念验证研究强烈表明，修复关节软骨的能力与对创伤后骨性关节炎的保护呈正相关。这一独特的遗传资源(即重组近交系)将被用来系统地识别有助于软骨再生的基因，从而促进年龄相关的原发骨关节炎和创伤后骨关节炎。用于实现这些特定目的的方法依赖于具有不同再生软骨和耳组织能力的重组近交系LG/J和SM/J的可用性，结合小鼠关节软骨损伤(全层)的手术技术和创伤后骨性关节炎模型(DMM-内侧半月板不稳定)。对软骨细胞和干细胞的功能分析将在极端愈合的菌株中进行。该项目的结果将是识别影响小鼠骨关节炎发育和关节软骨再生的基因，为探索人类基因组提供高质量的候选基因。这些重组近交系是由我的导师之一James Cheverud博士建立的，除了组织再生和长骨生长外，还被广泛用于研究其他复杂的疾病，如肥胖症和糖尿病。在过去的3.5年里，在合作中， 我们已经确定了其中一些菌株的表型，用于软骨再生和骨关节炎。假设：在重组近交系小鼠品系中可以确定基因变异，这些变异最终将与年龄相关的原发骨性关节炎和创伤后骨性关节炎的易感性和保护性相关。我们发展了一个假设，即再生关节软骨的能力与再生耳朵组织的能力在基因上是正相关的(Rai等人，关节炎大黄：2300-10)。2012年)。我们还开始研究关节软骨再生能力与骨关节炎易感性之间的关系，发现关节软骨再生与骨关节炎之间存在负相关(Hashimoto et al.，OsteoArcionyonage 20：562-71)。2012年)。我们还通过支链DNA技术分析了从8个菌株的组织切片中获得的组织裂解物上候选基因的表达。有几个基因的表达在菌株之间是显著可遗传的。代表DNA修复途径的四个基因(XRCC2，增殖细胞核抗原)和Wnt信号通路(Axin2，Wnt16)与这两种表型显著正相关，这表明组织愈合的共同遗传基础(在G3中审查)。这一建议的指导性假设是，可以确定参与软骨修复的基因，这些基因也将参与创伤后骨性关节炎。我们建议 通过基因定位、细胞功能分析以及识别与软骨修复和骨性关节炎相关的特定基因来跟进这些令人兴奋的结果。研究设计：本研究包括四个具体目标：(1)研究LGXSM重组近交系在原发和创伤后骨性关节炎关节软骨再生和发育中的表型差异。(2)定位青年和老年LGXSM重组近交系的遗传变异，定位所有性状的QTL，定位到10 cM的基因组间隔。(3)研究LGXSM重组近交系在原发和创伤后骨性关节炎关节软骨再生和发育中的分子差异。(4)监测精选LGXSM重组近交系软骨细胞和间充质干细胞的内在功能差异。结果的意义：我们将能够识别控制组织再生的基因或基因组(耳部创伤和膝关节软骨)，并防止患上与年龄相关的原发和创伤后骨性关节炎。在骨性关节炎中起作用的基因是未知的，在人类中的研究只针对原发骨性骨性关节炎，即使那样，也产生了很少的信息，主要是因为将会有许多基因的影响很小。这一限制可以通过我们的遗传小鼠模型来解决。最后，愈合者和非愈合者之间的细胞和分子差异将为软骨再生和去变形的机制提供新的见解。将这些发现应用于人类，有助于更好地对患者的OA进展风险进行分层，并确定可能的治疗干预目标。