质子感知受体OGR1介导胞内Ca2+水平调控椎间盘终板软骨细胞退变及其作用机制

The vertebral cartilage endplate (CEP) is the dominating passway of the nutrition for intervertebral disc (IVD). CEP degeneration, causing poor intervertebral disc cell nutrition, is one of the most important initial factors leading to IVD degeneration. 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. Pathological basis of nutrition reduction is ischemia and anoxia, similar with tissue acidosis. But it is not known if OGR1 is involved in the pathological process of IVD degeneration? In our previous study, we found that OGR1 was expressed in rat endplate chondrocytes. Knocking down OGR1 effectively decreased [Ca2+]i and attenuated acid-induced apoptosis of endplate chondrocytes. However, the role of OGR1 remains unclear in endplate chondrocyte degeneration through an acidosis-induced increase in [Ca2+]i. In this research, we plan to use OGR1 overexpression and OGR1 RNA interference to figure out the role of OGR1 in endplate chondrocyte degeneration ；the mechanism of OGR1 being involved in acid-induced calcium sensitive proteases and their downstream signal transduction will be studied; and the effect of blockage of OGR1 on CEP and IVD degeneration in vivo by specific blocker of OGR1 will be investigated. In general, the role of OGR1 involved in the apoptotic injury of the vertebral endplate chondrocytes and IVD degeneration may be clarified by these findings, based upon which the novel therapeutic strategies for IVD degeneration could be provided.

椎体软骨终板是椎间盘营养供应主要通道，软骨终板退变造成椎间盘细胞营养代谢障碍，是椎间盘退变始动因素之一。软骨终板的缺血缺氧与哮喘等炎症酸化具有相似病理基础，已证实质子感知受体OGR1在哮喘中发挥重要作用，其是否参与终板及椎间盘退变过程却还不得而知。我们前期研究发现终板软骨细胞表达OGR1，敲除OGR1可抑制胞内Ca2+升高，进而减少软骨细胞凋亡。但OGR1如何介导胞内Ca2+水平调控终板软骨细胞退变的作用机制尚不明确。本研究将采用：1) 用OGR1过表达和沉默以分析OGR1参与酸诱导的终板软骨细胞损伤与代谢的作用；2)分析OGR1介导酸诱导细胞损伤的Ca2+信号通路；3）通过阻断OGR1受体以观察大鼠腰椎间盘退变模型修复效果，在动物体内进一步验证其作用。本研究拟通过以上方法，初步阐明OGRl受体对终板软骨细胞凋亡性损伤影响及机制，对深刻认识OGR1功能及寻找抗椎间盘退变新靶点有重要意义。

本项目拟阐明OGR1（Ovarian cancer G-protein-coupled Receptor 1，OGR1）受体对终板软骨细胞凋亡性损伤影响及机制，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。首先分析大鼠终板软骨细中OGR1的表达水平，使用 OGR1 shRNA沉默OGR1表达分析OGR1对于酸环境下终板软骨细胞凋亡，细胞外基质以及金属蛋白酶的作用。其次通过共聚焦分析OGR1对软骨细胞内（[Ca2+]i）的影响，并通过一系列的阻断剂分析OGR1介导的酸引起的软骨细胞凋亡途径中的蛋白酶途径、CaMKⅡ途径以及具体的下游信号通路。最后在终板下注射30μl无水酒精造成终板软骨退变，通过阻断椎间盘的营养途径建立椎间盘退变模型，在术后1、2、3、4月通过组织学，影像学等水平进行验证分析，同时探索退下椎间盘的髓核组织中OGR1的表达水平。研究发现1、大鼠椎间盘的终板软骨细胞内有OGR1的高表达。椎间盘终板软骨细胞表达OGR1，受到细胞外酸化的调节；酸激活的OGR1在椎间盘终板软骨细胞凋亡和细胞代谢过程中发挥调节性的功能；2、OGR1介导的酸诱导软骨细胞凋亡是通过激活Ca2+敏感的蛋白酶和下游信号Bid、Bad、Caspase 3、PARP和caspase 3通路；3、无水酒精破坏终板下椎体的骨质微循环血运，导致终板退变引起椎间盘的营养障碍，最终髓核和纤维环也发生退变,此模型适合于椎间盘退变的病因学研究，OGR1在髓核细胞中的表达随着退变的加重而逐渐升高。本课题基本完成了预定的研究目标，并且已发表SCI科研论文11篇，科普文章2篇。