细胞骨架蛋白在机械力信号通路中的作用机制及其与椎间盘退行性变关系的研究

Disc degeneration is the primary cause of chronic low back pain. Due to the main function of the spine, mechanical stimuli play a pivotal role in the occurring and development of disc degeneration, however its detail mechanism, i.e. how mechanical stimuli induce cellular response, is largely unknown, especially in the intervertebral disc (IVD) cells. We hypotheses that cytoskeletal elements including actin, tubulin and vimentin are involved in the mechanotransduction pathways between the extracellular matrix (ECM) and cell nucleus in IVD cells, and its dysfunction will induce an abnormal metabolism of ECM components in IVD cells in response to mechanical stimuli. This ECM remodelling alter the composition of ECM and consequently change the mechanical distributions across the whole IVD, which will induce further metabolic imbalance within the ECM of IVD, increase the potential risk of a loss of tissue homeostasis and the likelihood of disc degeneration. To confirm our hypothesis, we will firstly make comparison in the organisation and expression of cytoskeletal elements including actin, tubulin and vimentin between normal and degenerative IVD tissues from patients, which provide us a direct evidence showing that cytoskeletal elements are involved in disc degeneration. Secondly, after disruption the individual organisation of cytoskeletal elements in IVD cells using different inhibitors, the alterations of ECM component metabolism in IVD cells in response to different mechanical stimuli are determined, which will help us to address the possibility that cytoskeletal elements are involved in the mechanotransduction pathways in IVD cells. Finally, the potential downstream signalling molecules involved in the cytoskeletal mechanotransduction pathways within IVD cells are screened using gene and antibody chips, which will show us the mechanism how cytoskeletal elements transform the mechanical stimuli applied on IVD cells into biological responses. These studies will provide detail information for the role of cytoskeletal mechanotransduction pathways contributing to disc degeneration occurring and development, increase our understanding for the mechanism of disc degeneration-induced chronic low back, and new strategies for its prevention and clinic therapies.

椎间盘突出是慢性腰背痛的主要诱因，但其病因机制不清。结合前期实验结果，我们提出椎间盘细胞中细胞骨架蛋白（actin,tubulin和vimentin）主导的机械信号传导通路参与椎间盘突出发生发展这一假说。通过对比人体正常和病变组织细胞中细胞骨架蛋白结构、分布和表达的差异，试图阐明其是否参与椎间盘突出的发生发展；利用抑制剂干扰不同细胞骨架蛋白的分布后，给予椎间盘细胞机械刺激，通过检测胞外基质成分代谢的改变，为证明细胞骨架蛋白参与人椎间盘细胞机械信号传导通路提供证据支持；利用抑制剂干扰细胞骨架蛋白的分布后，通过检测各个细胞骨架蛋白所主导的机械信号转导通路下游信号分子在不同机械刺激下的改变情况，探索细胞骨架蛋白参与机械信号传导通路的分子机制。本课题可以极大的增强我们对椎间盘突出发病机制的了解，为预防和治疗由椎间盘突出所引起的慢性腰背痛提供新的思路和证据支持，具有重要的理论和实践意义。

椎间盘突出是慢性腰背痛的主要诱因，但其病因机制不清。结合前期实验结果，我们提出椎间盘细胞中细胞骨架蛋白（actin,tubulin和vimentin）主导的机械信号传导通路参与椎间盘突出发生发展这一假说。通过对比人体正常和病变组织细胞中细胞骨架蛋白结构、分布和表达的差异，试图阐明其是否参与椎间盘突出的发生发展；利用抑制剂干扰不同细胞骨架蛋白的分布后，给予椎间盘细胞机械刺激，通过检测胞外基质成分代谢的改变，为证明细胞骨架蛋白参与人椎间盘细胞机械信号传导通路提供证据支持；本课题可以极大的增强我们对椎间盘突出发病机制的了解，为预防和治疗由椎间盘突出所引起的慢性腰背痛提供新的思路和证据支持，具有重要的理论和实践意义。.收集和保存人正常和病变椎间盘组织样品，检测细胞骨架蛋白及其相关蛋白在人正常和病变椎间盘组织(髓核和外纤维环)中的分布和表达，分离和体外培养人椎间盘髓核和外纤维环细胞，同时干扰actin在细胞内的分布，进行体外重复性压力和拉力刺激实验，利用免疫荧光染色、激光共聚焦扫描、real-time PCR以及Western blotting 等技术对比分析对照组和实验组之间的差异，已得出了主要结果，相关论文已经发表。