细胞骨架F-actin与L-型钙通道在骺板软骨细胞力学信号转导中的偶联及其作用

Mechanical loading have been extensively considered to be a crucial regulatory factor for growth plate cartilage and bone development, but its exact mechanisms of mechanotransduction are still not completely understood. In our previous studies, we have found that the F-actin microfilament of cytoskeleton and L-type calcium channel were both involved in the mechanotransduction of growth plate chondrocytes, and we hypothesized that the coupling of them play an important role in pathological growth plate. In our studies, firstly, the microscopic magnetic resonance elastography (μMRE) technique will be used to detect the deformation of growth plate chondrocytes under physiological and pathological load environment. After that, growth plate chondrocytes of the greatest deformation zone will be isolated and purified by magnetic activated cell sorting (MACS) or micro-dissection technique. Secondly, chondrocytes will be subject to desirable mechanical loading, accompanied with receptor antagonist or RNA interference to interrupt potential signaling pathways, and the relationship of possible signal transduction factors, such as integrins, cytoskeleton-related proteins, and intracellular calcium, will be analyzed. Thirdly, chondrocytes will be subject to excessive or overloaded mechanical loading, and cell apoptosis and apoptosis-related gene expression will be evaluated and possible mechanisms of inappropriate biomechanical environment resulting in growth plate dysplasia or damage will be explored. Quantitative characterization and basic knowledge of the underlying mechanisms involved in the mechanotransduction of growth plate will be helpful in developing an optimal strategy for interventions of pathological growth plate in early stage.

力学环境对骺板软骨细胞的功能活动及骨的发育具有重要影响，而其力学信号转导的具体机制以及相关转导因子的变化和相互联系尚不明确。申请者在前期的研究中发现细胞骨架微丝F-actin和L-型钙离子通道参与骺板软骨细胞的力学信号转导过程，推测二者之间可能存在偶联关系且在骺板应力相关性疾病的发病或病变进展中发挥重要作用。本项目拟采用显微磁共振弹性成像（μMRE）技术，观察骺板软骨各细胞层在应力下的形变情况；免疫磁珠/显微切割技术分离和体外培养应力主导层细胞并予以体外力学干预，观察和检测干预前后整合素、细胞骨架相关蛋白、胞内钙浓度等的变化及联系；运用拮抗/阻断剂或RNAi技术，研究可能的偶联关系；在此基础上，予以异常（超负荷）力学干预，探讨异常应力所致骺板软骨发育异常的机制。深入认识骺板软骨细胞力学信号转导过程和相关转导因子的作用及相互关系，可能为靶向干预骺板应力相关病变的发生或进展提供新思路。

力学刺激对骺板细胞的功能活动及骨的正常发育与成熟起着重要作用，但其力学响应（信号转导）的具体机制仍不清楚。细胞骨架F-actin和钙离子通道是重要的力学信号转导因子。本项目实施过程中，免疫磁珠分离并体外培养骺板软骨不同细胞层细胞；通过四点弯曲应力加载仪予以体外力学干预，观察和检测细胞干预前后细胞骨架、胞内Ca2+等的变化及骺板软骨生物学相关因子（PTHrP、Sox9等）；对可能的信号转导通路，予以拮抗/阻断剂或RNAi技术；予以超负荷力学干预，观察和检测应力过载下骺板软骨细胞自噬情况；开展初步的动物跑台实验，通过μMRI技术，观察活体骺板软骨在应力下的形变情况。.通过上述研究内容的开展，得出以下结果：1. 在应力作用下，以CD200为标记物免疫磁珠分选的肥大层和前肥大层细胞，其PTHrP的表达呈时间和强度依耐性；F-actin在应力作用下发生重构；破坏F-actin的完整性可抑制应力所致PTHrP表达升高。2. 应力可上调Sox9的表达水平；添加硝苯地平阻断钙离子通道可部分抑制应力作用下Sox9表达升高；激光共聚焦未能观察到钙波。3. 应力调控骺板软骨细胞YAP和ERK的表达呈时间和强度依耐性；破坏F-actin和阻断RhoA可抑制应力作用下YAP和ERK表达升高；阻断RhoA（C3毒素）和细胞骨架松弛素D可导致F-actin应力纤维形成障碍；细胞周期发生改变，S期及G2/M期比率增加，同时细胞周期调控因子（PCNA和cyclin D1）在应力作用下亦表达上升，通过siYAP及ERK阻断（U0126）可抑制上述变化。该结果提示RhoA、YAP和ERK在力学信号转导通路中扮演重要角色。4. 应力所致细胞凋亡因子LC-Ⅱ/Ⅰ比值呈时间和强度依耐性，应力过载（5500με）可导致LC-Ⅱ/Ⅰ比值显著增加；凋亡相关基因（LC3、Ulk1、Atg5、Becn1）发生改变；透射电镜结果显示细胞自噬泡数量增加，3-MA可阻断该变化；短时间应力过载不直接增加自噬和凋亡率，但能诱导自噬体形成，细胞骨架松弛素D可抑制上述改变。5.动物跑台实验结果显示骺板软骨的形态发生一定变化。.上述结果表明钙离子通道参与力学信号转导过程，且充分证实了细胞骨架F-actin在力学响应中的枢纽地位。同时，结果显示应力过载可导致细胞自噬状态的激活，解释超负荷/异常应力导致骺板发育异常的可能原因。