椎间盘退变中CK8介导的压力承载对髓核细胞命运及功能的调控研究

Extensive compression is one of the main predispositions that induce intervertebral disc degeneration (IDD). In the biomechanic environment, cytoskeletal protein is vital for cell morphology and signal transduction. Previously, we found that cytokeratin 8 (CK8) expression in human nucleus pulposus (NP) cells is closely related with IDD. Moreover, extensive compression can induce CK8 degradation and morphological changes of NP cells via phosphorylation. However, the regulatory mechanism of CK8 in mechanical force with regard to NP cell degeneration and IDD remains unknown. With the application of clinical specimens, animal models and in vitro cell culture system, as well as controlled load culture, virus transfection and cell co-cultures, the current study is aimed to elucidate the key role of CK8 in biological loading with regard to its involvement of Fas/FasL mediated apoptosis and HLA mediated immune escapes. Hence clarify the regulatory mechanism of CK8, which is an essential part of cytoskeletal protein, in biological loading and cell signal pathways. The implementation of this project will provide important evidence for IDD etiology as well as fundamental research and biological treatment strategies for IDD.

过度压力是导致椎间盘退变（IDD）发生的重要因素。细胞骨架蛋白作为细胞内离散网络结构，在力学影响细胞命运和多种功能中具有重要调节作用。课题组前期发现细胞角蛋白8 (CK8)表达与椎间盘的退变相关，并明确过度压力可通过磷酸化降解CK8表达，引起细胞形变。然而，CK8介导力学因素对髓核细胞的调控及启动IDD的分子机制仍不明确。本项目拟利用患者临床标本、动物模型及体外细胞模型构建，采用可控压力培养、病毒转染和共培养等实验技术，研究CK8在髓核细胞力学承载过程中参与Fas/FasL介导的细胞凋亡及HLA-I调控的免疫逃逸过程中的关键作用，从而阐明CK8作为细胞骨架蛋白核心分子介导髓核细胞力学调控的关键分子机制，深入了解IDD发病关键机制，并为以CK8为靶分子的相关基础研究及生物治疗奠定重要基础。

椎间盘退变是脊柱退行性疾病的重要病理基础。椎间盘由位于中央的髓核、外周包绕的纤维环和上下覆盖的软骨终版构成，髓核退变是椎间盘退变的关键病理环节。异常应力是髓核退变的独立致病因素。本项研究发现，细胞角蛋白8（CK8）在人椎间盘髓核特异性表达，CK8蛋白表达水平随人椎间盘退变程度加重而降低。髓核特异性Ck8敲除（Lepr-Cre;ck8fl/fl）加重异常应力致小鼠椎间盘退变。体外机制研究表明，异常应力增加髓核细胞内Ck8转录，髓核细胞内的CK8蛋白水平由增高的Ck8转录水平和髓核细胞内增高的蛋白酶体活性共同决定，体外敲低Ck8加重异常应力致髓核细胞退变和凋亡；体外过表达Ck8部分挽救异常应力致髓核细胞退变和凋亡，而通过抑制蛋白酶体途径恢复的CK8蛋白水平没有治疗效果；进一步研究表明：异常应力激活的RHOA-蛋白激酶N（PKN）磷酸化CK8蛋白43位丝氨酸，阻碍高尔基体蛋白RAB33B至ATG16L1胞内转运，自噬小体组装受阻，参与髓核细胞退变表型的进展和凋亡。体外敲低磷酸化转导通路关键激酶Pkn1/2部分挽救异常应力下的髓核细胞退变和凋亡。体内研究表明，退变模型早期过表达Ck8，敲低Pkn1/2均可缓解异常应力致椎间盘退变，而退变模型晚期只有敲低Pkn1/2才具有治疗效果。敲低Pkn1/2是治疗窗口更广的椎间盘退变潜在生物治疗靶点。.项目进展中我们还发现：炎症因子诱导下，髓核细胞miR-31a-3p、miR-384-5p、miR-582-5p表达显著增高，且靶向降低Ck8表达，导致CK8蛋白水平显著降低。同时，在异常应力与炎症模型下，降低的CK8蛋白均会调控免疫细胞对髓核细胞的识别杀伤。.课题组还应用蛋白组学技术全面描述异常应力致椎间盘退变的分子改变。我们首次报道线粒体蛋白SIRT5在退变髓核中显著下调。并在体内外、经多路径证明了Sirt5在髓核稳态中的重要作用。.综上，课题组在全面阐述CK8参与异常应力致椎间盘退变机制和干预措施的基础上，还应用多组学手段，从力学信号与分子信号偶联机制揭示髓核细胞的命运转归，并鉴定了多个潜在的椎间盘退变干预生物靶点。.