酸环境下OGR1受体介导髓核细胞自噬调控椎间盘退变及其机制研究

Disc degeneration is the pathological basis of spinal degeneration disease, mainly characteried by the decrease of nucleus pulposus cells and disfunction, caused by the microenvironment associated with tissue acidosis. Interestingly, several recent studies have shown that proton-sensing receptor OGR1 plays an important role in the pathologic process of inflammation associated with tissue acidosis such as asthma. Pathologic basis of disc degeneration is ischamia and anoxia, similar with tissue acidosis. In our previous sutdy, we sound that OGR1 was expressed in rat nucleus pulposus cells, and the acid microenvironment can promote the expression of autophagy in nucleus pulposus cells. But it is not known if OGR1 is involved in the pathological process of disc degeneration? And the relationship between autophagy of nucleus pulposus cell and acid microenvironment is still unknown. In this research, we plan to use OGR1 overexpression and OGR1 RNA interference to figure out the role of OGR1 in autophagy of nucleus pulposus cell in acid microebvironment and its effect on disc degeneration; the mechanism of OGR1 being involved in acid-induced calcium sensitive proteases and their downstream signal transduction will be studied. In general, the role of OGR1 involved in the autophagy of nucleus pulposus cell and disc degeneration may be clarified by these findings, based upon which the novel therapeutic strategies for disc degeneration disease could be provided.

椎间盘退变是脊柱退变性疾病的病理基础，其核心是髓核细胞数量减少和功能降低。缺血缺氧和组织酸化的微环境是导致椎间盘退变的重要因素。质子感知OGR1受体是细胞膜上的酸受体之一，在哮喘、炎症等酸化过程中发挥重要作用，而椎间盘的缺血缺氧与酸化与上述病变酸化过程具有相似病理基础。我们在前期研究中已发现髓核细胞表达OGR1受体，且酸环境可促进髓核细胞自噬表达。但酸环境如何调控髓核细胞自噬，质子感知OGR1受体如何参与其中，最终对椎间盘退变产生什么影响，这些尚不得而知。我们提出假设：酸环境下OGR1介导的髓核细胞自噬在椎间盘退变过程中起关键作用。为证实这一假设，本课题拟用：1）OGR1过表达和沉默以分析OGR1参与酸环境下椎间盘髓核细胞的自噬作用；2）分析OGR1介导酸环境下髓核细胞的自噬的信号通路以验证其对细胞功能和代谢的影响，阐明OGR1受体对髓核细胞自噬的影响及机制，为脊柱退变疾病治疗寻求新思路。

本项目拟阐明质子感知受体OGR1（Ovarian cancer G-protein-coupled Receptor 1，OGR1）是否介导酸环境下椎间盘髓核细胞的自噬及其在椎间盘退变中的作用，为深刻理解OGR1的功能提供理论基础并为临床抗椎间盘退变提供潜在的新靶点。收集临床就诊患者髓核组织行HE染色和免疫组化观察髓核细胞中OGR1的表达水平。培养大鼠正常髓核细胞，不同PH培养基处理，免疫荧光，Western blotting以及藻酸钠凝胶三维培养后的免疫组化观察细胞中OGR1表达与微环境中酸的关系。AO染色，SQSTM1/p62，LC3-II的Western blotting, mRFP-GFP-LC3转染，透射电镜(Transmission Electron Microscope，TEM)以及巴弗洛霉素A1（Bafilomycin A1)的使用观察不同PH培养基中髓核细胞中自噬和自噬流的水平以及自噬泡数量的大小。构建OGR1-shRNA慢病毒载体转染髓核细胞沉默OGR1表达并进行验证，再使用上述的方法观察髓核细胞自噬和自噬流表达。最后使用II型胶原的免疫荧光和Alcian染色观察OGR1沉默后髓核细胞在酸环境中的功能和活力。OGR1免疫组化结果显示，在退变的髓核组织中OGR1的表达明显升高。平面培养的和三维培养的髓核细胞中，随着细胞外培养基PH值的降低，OGR1的表达升高，并且与PH值的改变具有一定的依懒性。酸环境促进髓核细胞中酸性溶酶体的数量和LC3-II表达明显升高。Western blotting 显示PH6.4，作用时间24-48小时的时候，细胞中LC3-II的表达量相对最高。mRFP-GFP-LC3显示酸刺激下髓核细胞中自噬体到自噬溶酶体的转变，使用Bafilomycin A1干预后明显增加胞浆中大量自噬体的堆积，而LC3-II的表达相对纯酸刺激进一步的升高。TEM显示PH6.4时细胞中的自噬体和自噬溶酶体的数量明显增多，甚至与雷帕霉素作用时的自噬体数量类似。OGR1-shRNA1沉默OGR1表达的效果明显。OGR1沉默后逆转酸诱导的LC3-II水平的升高和自噬体数量的增加，并且导致细胞中SQSTM1/p62的积聚。mRFP-GFP-LC3共聚焦显示OGR1沉默后细胞中自噬体和自噬溶酶体数量均减少。髓核细胞的功能方面，酸明显抑制髓核细胞表达II型胶原