痤疮丙酸杆菌(P.acnes)通过Dlx-2负调控Lubricin参与Modic改变的作用及机制研究

Modic changes (MCs) is an important sign of endplate degeneration in the lumbar spine. Although its pathogenesis is unclear, it is thought to be associated with Propionibacterium Acnes (P.acnes) infection. Our previous studies confirmed that P.acnes can result in MCs by injecting P.acnes to rabbit endplates. Lubricin, a mucinous glycoprotein secreted by articular chondrocytes, is proved to have anti-inflammatory effects in the progression of osteoarthritis. However, whether lubricin is expressed in the vertebral endplate and its potential function remain unknown, especially in P.acnes induced MCs. Our prelimary research revealed the expression of Lubricin in chondrocytes of vertebral endplate, and the expression level of Lubricin was diminished in the presence of MCs. Further, culturing endplate chondrocytes with the presence of P.acnes supernate showed the expression level of Lubricin was significantly suppressed, as well as the expression of Dlx-2. Interestingly, we found that the promoter of Lubricin contains the binding site of Dlx-2 by JASPAR prediction. Thus, our hypothesis is that P.acnes can aggravate MCs and endplate degeneration by inhibition of Lubricin expression via Dlx-2. This study will focus on assessing the protection effect of Lubricin in P.acnes induced MCs by means of gene overexpression/knockdown, or exogenous Lubricin supplementation. Also, inhibitory effect of P.acnes in Lubricin expression in endplate and its regulating mechanism with the mediation of Dlx-2 will be studied. Based on our research, deeper understanding of lumbar spinal endplate changes will be achieved, providing theoretical support in clinical diagnose and therapy of patients afflicted with related conditions.

Modic改变是腰椎终板退变的重要征象，目前认为P.acnes感染是其发生的重要机制之一，申请人团队也通过新西兰大白兔终板注射P.acnes成功诱导了Modic改变的发生。研究表明，关节软骨表达Lubricin，可抑制骨关节炎的进展，而其在终板软骨中的表达和功能尚不清楚。我们前期研究发现终板存在Lubricin表达，且当发生Modic改变时显著降低；P.acnes培养上清液刺激终板软骨细胞后，其表达亦减少，并检测到Dlx-2同步下调；通过JASPAR网站预测发现Lubricin启动子区域含有Dlx-2的结合位点，进而推测P.acnes可能通过Dlx-2负调控Lubricin参与Modic改变。因此，我们拟通过基因过表达/敲低以及补充外源性Lubricin，研究其在Modic改变中的保护作用，并进一步探索P.acnes对终板Lubricin表达的抑制作用及其中Dlx-2介导的调控机制。

Modic改变是腰椎终板退变的重要征象之一，目前认为痤疮丙酸杆菌（P.acnes）感染是导致其发生的重要机制之一。申请人团队也通过新西兰白兔终板内注射P. acnes成功诱发了Modic改变的发生。近年来研究表明，关节软骨细胞可表达Lubricin，抑制骨关节炎的进展，而其在椎体终板软骨细胞中的表达和功能尚不清楚，尤其在P. acnes诱导的终板Modic改变中的作用暂无报道。我们前期研究发现腰椎终板中存在Lubricin表达，在发生Modic改变的终板中其表达显著减少。同时，P.acnes培养上清液刺激终板软骨细胞后，亦可发现Lubricin表达显著减少，并检测到Dlx-2的同步下调。我们推测P．ances可能通过Dlx-2负调控Lubricin，参与Modic改变，因此我们选取2014年9月至2017年2月我院腰椎椎间融合和腰椎骨折前路手术的终板标本，通过免疫组化发现Lubricin在椎体终板软骨细胞中表达，并提取mRNA，进行qPCR检测，发现发生Modic改变的终板Lubricin表达显著减少。取Modic改变动物模型新西兰白兔终板行免疫组化，发现Lubricin在新西兰白兔终板内有表达，其中软骨终板较骨性终板表达较高，同时发现有Modic改变的终板内Lubricin表达显著减少。并提取mRNA，进行qPCR检测，进一步证实发生Modic改变的终板Lubricin表达显著减少。分离培养3月龄新西兰白兔终板软骨细胞，并与不同浓度P.acnes细菌上清液共培养48h，提取终板软骨细胞mRNA，进行qPCR检测发现终板合成代谢相关基因（Aggrecan、Col-II、Sox-9）表达下降，终板分解代谢相关基因（MMP-1、ADAMTS-5）表达上调，Lubricin经P.acnes细菌上清液刺激后表达量明显下降。以此证明椎体终板软骨细胞中存在Lubricin的表达，而且发生Modic改变的终板Lubricin表达显著减少，Lubricin可能在Modic改变的发生过程中起抗炎和保护作用。此研究深化我们对临床腰椎终板退变的认识，为腰椎疾患的临床诊治提供理论支持。