生物钟基因CLOCK/BMAL1调控髓核细胞自噬节律性在椎间盘退变中的作用及机制研究

The circadian rhythms controls several basic physiological processes in our bodies. The previous researches have shown that the differences of spinal loading during the day and night lead to the circadian rhythms of pressure loaded on the nucleus pulposus tissue, as well as the microenvironment around the nucleus pulposus cells. The dysregulation of the circadian rhythms led to intervertebral disc degeneration(IDD). In our previous studies, we have demonstrated the protective role of autophagy in the adaptation of nucleus pulposus cells to the micro-environments, however the effect of autophagy rhythm in the IDD remains unclear. Our preliminary experiments found the circadian rhythms of autophagy-related proteins in the upright-posture rat model. The loading pressure activated autophagy and upregulated the expression level of Clock gene. On the other hand, silencing the Clock gene inhibited the activation of autophagy. Furthermore, over-expression of BMAL1 suppressed the phosphorylation of S6K1, a down-stream protein of mTOR. Given that, we hypothesize that CLOCK/BMAL1 regulates the level and rhythm of autophagy in nucleus pulposus cells through the mTOR pathway, which enable the cells to adapt the diurnal rhythm of loading pressure, to be free from apoptosis and dysfunction, integrated to protect the IDD. This program is established from the autophagy rhythm to clarify: 1) the effect and mechanism of compression on the rhythm of CLOCK/ BMAL1, 2) the mechanism of CLOCK/ BMAL1 in regulating the autophagy rhythm, and the role of autophagy rhythm of nucleus pulposus cells in adapting the changing of loading pressure. We would like to use RNAi, over-expression and animal models to provide some new theoretical basis and strategies for preventing the IDD.

昼夜节律调控机体正常生理过程。研究报道脊柱载荷的昼夜不同导致髓核组织压力及微环境的节律性，节律破坏引起椎间盘退变。我们前期工作证实自噬在髓核细胞适应微环境中的保护作用，但是自噬节律性在椎间盘退变中的作用尚不清楚。预实验在直立大鼠椎间盘模型中发现自噬的昼夜节律性，压力载荷下髓核细胞自噬激活及钟基因CLOCK表达上调，沉默CLOCK抑制自噬水平，过表达CLOCK伴侣BMAL1抑制mTOR下游S6K1磷酸化，我们假设压力载荷下CLOCK/BMAL1通过mTOR通路调控髓核细胞自噬水平及节律性，适应压力昼夜改变防止细胞凋亡和功能紊乱缓解椎间盘退变。本项目从自噬节律性出发，明确1）压力载荷调控CLOCK/BMAL1节律性的作用及机制；2）钟基因介导自噬水平和节律改变提高髓核细胞适应能力在椎间盘退变中的作用及机制。通过RNAi，过表达转染以及动物模型验证，为椎间盘退变的防治提供新的理论基础和防治策略。

本项目拟阐明生物钟基金CLCOK/BMAL1介导异常载荷对髓核细胞自噬和功能的影响，为深刻理解生物节律对椎间盘退变的作用并为临床抗椎间盘退变提供潜在的新靶点。首先分析生物钟BMAL1，CLOCK在髓核细胞内地分布及表达，生物钟基因的振荡节律与年龄的关系；通过不同浓度的D-gal诱导髓核细胞衰老，进一步分析细胞衰老对生物钟的影响。其次通过牵张系统以18% 0.2Hz的正选波循环牵拉（CTS）原代髓核细胞，分析髓核细胞生物钟基因以及功能基因的变化；通过质粒转染过表达BMAL1，分析髓核细胞功能的变化；通过latrunculin B和cytochalasin D破坏细胞骨架，Y-27632和siRHOA抑制RHO/ROCK通路分析其所起的作用。最后构建直立大鼠模型，通过改变光照/黑暗的周期时间构建节律紊乱大鼠模型。核磁共振，X光片，组织学染色证实髓核组织的退变。Western blotting，qRT-PCR分析生物钟核心基因的表达水平。研究发现1、不同年龄大鼠显示其生物钟基因表达节律具有差异性，24月龄的老年大鼠其节律周期比年轻大鼠要长，而振荡幅度降低，节律相位也发生改变。衰老的髓核细胞节律紊乱，振荡幅度降低；2、过度的CTS载荷改变BMAL1和CLOCK的振荡周期及振幅，并且无法被地塞米松恢复。同时髓核细胞在18％CTS 0.2Hz循环拉伸24、36和48小时后，细胞外基质合成减少而且降解增多；BMAL1过表达能够部分缓解CTS对NP细胞的影响。Rho/ROCK信号通路可能介导异常载荷对生物钟基因的影响。3、长时间的站立干扰髓核组织内在的生物节律，减少了髓核组织中生物钟基金BMAL1和CLOCK的表达，这不仅促进正常生物节律大鼠椎间盘的退变，而且进一步加快了昼夜颠倒椎间盘的退变。本课题基本完成了预定的研究目标，在课题的资助下已发表SCI科研论文10篇，单篇IF最高为16.8分，获得上海市人才发展基金，上海市医苑新星等人才基金资助。