Meniscus fibrochondrocytes mechano-hypoxia transduction

半月板纤维软骨细胞机械缺氧转导

• 批准号: RGPIN-2018-06290
• 负责人: Adesida, Adetola
• 金额: $ 6.85万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=765811
• 关键词: Meniscus; fibrochondrocytes; mechano; hypoxia; transduction

Meniscus fibrochondrocytes (MFCs) are embedded in a collagen rich and proteoglycan constitutive extracellular matrix (ECM), which they synthesize and exist within a mechanically active and low oxygen tension (hypoxia; 1-7% O2) microenvironment within the knee. But there is no fundamental understanding on how MFCs simultaneously receive and decipher mechanical loads and hypoxic stimuli. This proposal aims to identify and characterize how MFCs simultaneously respond to mechanical and hypoxic stimuli within MFCs synthesized ECM. The findings of this research program will create new knowledge on MFC cell physiology as well as create a physiologically relevant model system to study the response MFCs to combined mechanical loads and hypoxia. We have demonstrated that the transcription factor, hypoxia inducible factor 1 (HIF-1), plays a significant role in the response of MFCs to hypoxia (5%O2). Moreover, hypoxia enhanced the matrix-forming phenotype of MFCs. Other investigators have reported that hypoxia increased the mechanical characteristics of ECM embedding articular chondrocytes after in vitro culture. To this end, hypoxia signaling may be of critical importance in the physiology of MFCs within a mechanically active knee. However, the concerted interplay of mechanical loads and hypoxia on MFCs' matrix-forming phenotype is yet to be explored or understood at both the transcriptional and translational level. We will investigate the simultaneous interplay of mechanical and hypoxia signaling pathways in MFCs embedded in MFCs synthesized matrix through the combination of tissue engineering strategies, transcriptome profiling and use of pathway-focused gene expression PCR array technology. Pathway-focused PCR array will enable us to narrow down and identify genes that are simultaneously modulated under combined mechanical loads and hypoxia. We will combine the capabilities of our newly acquired Canadian Foundation for Innovation (CFI) funded TA Instruments mechanical loading bioreactor and a Biospherix Xvivo X3 hypoxia workstation to explore the molecular pathways associated with combined of mechanical load and hypoxia in MFCs embedded in tissue engineered ECM formed from in vitro culture of MFCs. Post-experiment analyses will include transcriptomic analysis, qPCR, protein arrays, immunofluorescence, histology and biochemical assays. The planned experiments will ultimately create new knowledge in cell physiology and natural sciences by determining the transcriptional and translational outcome of combined mechanical loads and hypoxia on MFCs, and may prove significant to the fields of mechanobiology.

半月板纤维软骨细胞（MFC）包埋在富含胶原和蛋白聚糖的组成性细胞外基质（ECM）中，它们合成并存在于膝关节内的机械活性和低氧张力（缺氧; 1- 7%O2）微环境中。但是，对于MFC如何同时接收和破译机械负荷和缺氧刺激还没有基本的了解。该建议旨在确定和表征MFC如何同时响应于MFC合成ECM内的机械和缺氧刺激。这项研究计划的结果将创造MFC细胞生理学的新知识，并创建一个生理相关的模型系统，以研究MFC对机械负荷和缺氧的反应。我们已经证明，转录因子，缺氧诱导因子1（HIF-1），发挥了重要作用，在MFC的响应缺氧（5%O2）。此外，缺氧增强了MFC的基质形成表型。其他研究者报道，缺氧增加了体外培养后包埋关节软骨细胞的ECM的机械特性。为此，缺氧信号可能是至关重要的生理学中的MFC内的机械活动的膝盖。然而，机械负荷和缺氧对MFC基质形成表型的协同相互作用还有待于在转录和翻译水平上探索或理解。我们将通过结合组织工程策略、转录组分析和使用聚焦于通路的基因表达PCR阵列技术，研究嵌入MFC合成基质中的MFC中机械和缺氧信号通路的同时相互作用。聚焦于通路的PCR阵列将使我们能够缩小范围并识别在机械负荷和缺氧联合作用下同时受到调节的基因。我们将联合收割机结合我们新收购的加拿大创新基金会（CFI）资助的TA仪器机械负载生物反应器和Biospherix Xvivo X3缺氧工作站的能力，探索与嵌入MFC中的机械负载和缺氧相结合的分子途径。组织工程化ECM由MFC体外培养形成。实验后分析将包括转录组学分析、qPCR、蛋白质阵列、免疫荧光、组织学和生化测定。计划中的实验将通过确定MFC上组合机械负荷和缺氧的转录和翻译结果，最终在细胞生理学和自然科学中创造新的知识，并可能证明对机械生物学领域具有重要意义。