自噬调控外泌体/miR-27a分泌促进不同髓核细胞之间“对话”在抑制基质降解中的作用机制

The inflammation of the peripheral nucleus pulposus and degradation of extracellular matrix (ECM) caused by the ingrowth of nerve and blood vessel are the important factors of IDD. Our previous work have proved that autophagic activation maintains nucleus pulposus cell (NPCs) vitality and metabolic balance of ECM, however, the detailed regulatory mechanism is not clear. The previous research has proved the association between autophagy and exosomes in chondrocyte, but whether autophagy could regulate exosomes in nucleus pulposus cells remains unclear. Our preliminary experiments found that the autophagic activation could promote the exosome secretion from normal NPCs, and the exosomes could inhibit the degradation of the ECM of NPCs under inflammation. In addition, high-throughput chip found that the miR-27a was significantly increased in the exosomes from normal NPCs, and bioinformatical analysis showed that MMP-13 might be the target gene of miR-27a, suggesting that autophagy might inhibit the ECM degradation by promoting exosome secretion and releasing miR-27a which entering the nearby NPCs under inflammation and inhibiting the production of MMP-13. In order to prove the above hypothesis, We focus on the association between autophagy and exosomes, and intend to clarify the 3 following problems:1) the effect and mechanism of autophagy on exosome secretion; 2) the inhibitory role and mechanism of exosome and miR-27a on the degradation of ECM in NPCs under inflammation; 3) the effect of these factors o n IDD. Our study will help in exploring a new mechanism of disc degeneration and in proving the scientific evidence and new target in treatment of disc degeneration by the RNAi technology, over-expression transfection, and the injection of exosomes to the discs in vivo.

神经血管长入引起周围髓核组织炎症反应及细胞外基质（ECM）降解是椎间盘退变的重要因素。课题组前期工作证实自噬缓解髓核ECM降解，但调控机制尚不清楚。研究发现自噬调节软骨细胞外泌体（exosomes）形成，髓核与软骨相似，自噬是否可以通过外泌体影响髓核ECM的降解？预实验发现激活自噬促进正常髓核细胞分泌外泌体，提取外泌体抑制炎症环境下髓核细胞ECM降解，高通量芯片发现外泌体高表达miR-27a，生物信息学分析MMP-13为靶基因，提示自噬可能促进正常髓核细胞分泌外泌体，携带miR-27a等miRNA作用周围的炎症髓核细胞中MMP-13抑制ECM降解。本项目从自噬与外泌体关系出发，明确1）自噬促进外泌体miR-27a分泌及机制，2）外泌体携带miR-27a抑制炎症髓核ECM降解及机制。通过RNAi，过表达转染以及动物模型外泌体miR-27a注射，为椎间盘退变的防治提供的新的理论基础和防治策略。

神经血管长入引起周围髓核组织炎症反应及细胞外基质（ECM）降解是椎间盘退变的重要因素。课题组前期工作证实自噬缓解髓核ECM降解，但调控机制尚不清楚。预实验发现激活自噬促进正常髓核细胞分泌外泌体，提取外泌体抑制炎症环境下髓核细胞ECM降解，高通量芯片发现外泌体高表达miR-27a，生物信息学分析MMP-13为靶基因，提示自噬可能促进正常髓核细胞分泌外泌体，携带miR-27a等miRNA作用周围的炎症髓核细胞中MMP-13抑制ECM降解。为明确这一机制，我们进行了一系列研究。我们发现：1）使用自噬激活剂雷帕霉素（rapamycin）刺激髓核细胞可以促进髓核细胞外泌体的释放，而使用自噬抑制剂巴弗洛霉素A1（bafilomycin A1）以及利用ATG5 siRNA沉默自噬相关基因抑制自噬则会抑制髓核细胞外泌体的释放。并且通过Western blot、PCR、免疫荧光等技术证实自噬通过激活RhoC/ROCK2通路促进髓核细胞外泌体的释放；2）雷帕霉素激活髓核细胞自噬上调髓核细胞外泌体中miR-27a的水平，并且通过共聚焦显微镜拍摄荧光标记的外泌体证实其可被髓核细胞胞质中。进一步Western blot、PCR、免疫荧光证实激活自噬促进髓核细胞分泌的外泌体miR-27a可以通过抑制MMP-13从而降低炎症微环境下髓核细胞细胞外基质包括Aggrecan、Collagen II的降解。3）动物实验提示，椎间盘内注射外泌体/miR-27a（100ul）可以减轻针刺退变造模的SD大鼠椎间盘退变程度，表明其对于椎间盘退变具有保护作用。以上实验数据表明，自噬通过激活RhoC/ROCK2通路促进髓核细胞外泌体/miR-27a的分泌，并且外泌体/miR-27a可以通过抑制MMP-13从而减轻炎症下髓核细胞ECM降解，减轻椎间盘退变。本研究为椎间盘退变的防治提供了新的理论基础。