FAM38A基因调控机械敏感离子通道Piezo1与分子轨道线性马达交互作用在OA发病中的关键分子机制

The common characteristics of the sporty, traumatic, inflammatory and degenerative joint diseases are the wear or erosion of the articular cartilage, which are the main reasons for pain and disability of extremities. Clarify the key molecular mechanism of abnormal mechanical stress for pathogenesis of osteoarthritis has significant importance for control and prevention of this disease as well as finding new target for treatment. Piezo1 is a newfound ion channel protein closely related with mechanical stress on cell, and the FAM38A is the gene regulate the Piezo1. In order to find out the microscopic molecular mechanism of the role of Piezo1 plays in morphologic changing and apoptosis of chondrocytes, as well as the interaction with cytoskeleton, this project focuses on the connection between Piezo1 under multi-channel stretch stress with key linear molecular motor KIF C1 and KIF 18A, and influence on the molecular pathway of microfilament, microtubule and F-actin in chondrocytes, so as to discover the function of the cross-talk between Piezo1 and cytoskeleton for material transportation and energy conversion. By technologies like fluorescence resonance energy transfer, patch clamp and gene gun, etc, the activation of downstream key signal pathway like Rho A/PKN/ROCK, ERK5/MAPK and NF-κB are detected, so as to find the molecular mechanism for Piezo1 to regulate the morphologic changing and apoptosis of chondrocytes. Using the FAM38A（Piezo1）siRNA gene silencing/ hIGF-1 cDNA inducing bidirectional regulatory mode, osteoarthritis model in machin will be treated, to protect the chondrocytes and inhibit degenerative changing. By this means, new treatment strategy with important value for subsequent reasonable control of osteoarthritis and modified biological response will be found.

关节创伤与退变的共同特点是异常应力导致的关节软骨磨损，明确机械应力异常在OA发病的分子机制对于疾病防控及新治疗靶点发现有重要意义。Piezo1是新型机械敏感离子通道蛋白，FAM38A为其调控基因。为明晰其在软骨细胞形态变化及凋亡中作用的分子机制及与细胞骨架交互作用，基于前期工作基础，本课题研究多通道应力加载下Piezo1和线性马达KIF C1、KIF 18A变化的联系，和对分子轨道微丝、微管、F-actin架构的影响，发现对物质运输、能量转换的物理基础；利用FRET、膜片钳、基因枪等技术，探索Piezo1激活对信号通路Rho A/PKN/ROCK；ERK5/MAPK；NF-κB的影响，寻找Piezo1调控细胞形态变化及凋亡的分子机制；利用FAM38AsiRNA沉默及hIGF-1基因诱导双相干预食蟹猴OA模型，产生保护软骨细胞、抑制退变的新靶点，对OA的防控、生物学应答改良产生重要指导意义。

关节创伤与退变的共同特点是异常应力导致的关节软骨磨损，明确机械牵张异常在OA发病的分子机制对于疾病防控有重要意义。本研究从体外和体内两个层面分析力学刺激对新型机械敏感离子通道蛋白Piezo1介导的软骨细胞凋亡的信号通路的影响，同时对基因沉默/诱导双相调节干预后对靶细胞分子轨道、线性马达的影响，以及可能的信号转导机制进行监测。本研究构建的Piezo1-siRNA和hIGF-1基因慢病毒载体，可使软骨细胞中Piezo1表达减少hIGF-1表达增多。并发现沉默Piezo1和诱导hIGF-1可有效延缓加力引起的软骨细胞凋亡增多、细胞增殖减弱及炎性因子分泌。同时本研究对软骨细胞中Piezo1的离子通道特性进行了检测，发现沉默Piezo1和诱导hIGF-1对Piezo1通道特性（激活时间、失活时间和通道电流）无显著影响。通过激光共聚焦显微镜观察沉默Piezo1和诱导hIGF-1对细胞骨架的影响，发现随加力时间的延长，细胞骨架呈渐进性增粗及浓集，双相调节干预可减弱加力引起的细胞骨架变化。分子马达KIFC1及KIF18A与细胞骨架的变化密切相关，机械牵张促进了KIFC1及KIF18A的表达，且Piezo1沉默/hIGF-1诱导可显著减少加力所致的KIFC1及KIF18A的表达变化，以上结果提示Piezo1是通过KIFC1及KIF18A调控细胞骨架的变化进而影响细胞功能。本研究进一步通过激酶阻断实验及激光共聚焦显微镜等实验证实Rho A/PKN/ROCK、ERK5/MAPK和NF- κB途径作为Piezo1的下游途径介导力学刺激对细胞骨架及分子马达的调控。最后通过建立食蟹猴OA模型，证实双相基因干预模式，可特异性阻断 Piezo1 蛋白，产生保护软骨细胞、抑制退变、促进基质合成的效果。本研究结果将对OA的防控、生物学应答改良产生重要指导意义。