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: 8700992
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8700992
• 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; 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; Patients; Phase; Phenotype; Population; Population Genetics; Predisposition; Property; RNA; Recombinant Inbred Strain; Recombinants; Recruitment Activity; Research Design; Research Personnel; Resistance; Rheum; Risk; 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 development; cartilage regeneration; cartilage repair; follow-up; genetic resource; genetic strain; genetic variant; healing; insight; juvenile animal; laser capture microdissection; long bone; mouse model; novel; public health relevance; 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）：遗传和分子对软骨再生、原发性和创伤后骨关节炎的见解背景和原理：本提案来自M。Farooq Rai，软骨生物学和骨关节炎（OA）领域的基础科学研究员。指导阶段将在以下人员的指导下完成：i）琳达·J·桑德尔博士，矫形外科和细胞生物学系的软骨生物学家，ii）詹姆斯·M·M。Cheverud博士，解剖学和神经生物学系的群体遗传学家和形态学家，iii）Mathew J. Silva博士，矫形外科系的生物力学工程师和骨生物学家，以及iv）Ingrid Borecki博士， 统计基因组学部门。使用与“超级愈合”小鼠相关的近交系，我们已经定义了耳创伤愈合和关节软骨再生的表型，并且已经建立了这两种表型之间的强正相关性。此外，概念验证研究强烈表明，愈合关节软骨的能力与创伤后OA的保护呈正相关。这种独特的遗传资源（即重组近交系）将用于系统地鉴定有助于软骨再生的基因，从而有助于年龄相关的原发性OA和创伤后OA。用于实现这些特定目标的方法依赖于具有不同能力的重组近交系LG/J和SM/J的可用性，以再生软骨和耳组织，结合用于关节软骨损伤（全厚度）的小鼠手术技术和创伤后OA模型，内侧半月板的不稳定（DMM -OA模型）。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。该项目的成果将是鉴定影响小鼠OA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一位导师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疾病，如肥胖和糖尿病。在过去的3.5年里，在合作中， 我们已经建立了这些菌株中的一些菌株的软骨再生和OA的表型。假设：基因变异可以在重组近交系小鼠品系中鉴定，其最终与年龄相关的原发性OA和创伤后OA的易感性和保护相关。我们已经发展了这样的假设，即再生关节软骨的能力与再生耳组织的能力在遗传上正相关（Rai等人，关节炎大黄64：2300-10。2012年）。我们还开始研究关节软骨再生能力与OA易感性之间的关系，发现关节软骨再生与OA之间存在负相关性（Hashimoto et al.，骨关节炎Carnival 20：562-71. 2012年）。我们还分析了候选基因的表达，通过支链DNA技术对组织裂解物从组织切片的8个菌株。几个基因的表达是显着的菌株之间的遗传。代表DNA修复（Xrcc 2，Pcna）和Wnt信号传导（Axin 2，Wnt 16）途径的四个基因与两种表型显著正相关，表明组织愈合的共同遗传基础（在G3中审查）。该建议的指导假设是，可以鉴定出参与软骨修复的基因，这些基因也将参与创伤后OA。我们建议 我们将通过基因定位、细胞功能分析以及软骨修复和骨关节炎相关的特异性基因的鉴定来跟踪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）确定青年和老年LGXSM重组近交系在关节软骨再生和原发性OA和创伤后OA发展方面的表型差异。(2)在年轻和年老的LGXSM重组近交系中绘制关节软骨再生和原发性OA和创伤后OA发展的遗传变异图，将所有性状的QTL绘制到10 cM基因组间隔。(3)阐明LGXSM重组近交系在关节软骨再生和原发性OA和创伤后OA发展中的分子差异。(4)监测选定LGXSM重组近交系中软骨细胞和间充质干细胞的内在功能差异。成果的意义：我们将能够确定基因或基因组，管理组织再生（耳创伤和膝关节软骨）和保护免受年龄相关的原发性和创伤后OA。在OA中起作用的基因是未知的，并且在人类中的研究仅涉及原发性OA，即使这样，也没有产生什么信息，主要是因为有许多基因具有小的影响。这种限制可以通过我们的遗传小鼠模型来解决。最后，愈合者和非愈合者菌株之间的细胞和分子差异将提供新的见解软骨再生和去胚的机制。应用于人类，这些研究结果可以促进更好地分层患者的风险进展的OA，并确定可能的治疗干预的目标。