环形RNA在退变性颈椎间盘纤维环成纤维细胞成骨化生过程中的作用、机制及诊断标志物研究

Osteophytes are one of the reasons causing clinical symptoms related to vascular and nerve compression in cervical spondylosis. Thus, it’s reasonable to investigate the mechanisms of osteophyte formation. As recent studies show, annulus fibrosus fibroblasts from intervertebral disc are able to differentiate into cartilage and/or fibrocartilage cells, osteoblasts, and so on, which may be considered as potential cell resources for the formation of the tissues. And circular RNAs are a recently rediscovered new class of noncoding RNAs, which have regulatory potential of coding sequences. They have correlations to certain degenerative diseases like Alzheimer’s disease and specific expression profiling of the degenerative annulus fibrosus fibroblasts in our previous study, some of which may contribute to cell growth and differentiation. So we hypothesize that circular RNAs can be involved in cell differentiation process. Based on our previous study on the mechanisms of their biogenesis and cervical spondylosis, we intend to screen and analyze the expression profiles of circular RNAs in the annulus fibrosus fibroblasts, and study the effects that circular RNAs take on the osteogenesis course. Then, the mechanisms will be further explored related to osteophyte formation. Beside, circular RNAs have relative stable structures resistant to RNase R digestion and can be detected in the plasma, indicating them potential possibilities to serve as candidate biomarkers. Therefore, we intend to compare the expression patterns of circular RNAs in the plasma ,between the health people and patients with different severities, via sequencing and computational analysis. After screening specific and sensitive candidates, we will test and verify the molecules among models of different osteogenic induced models and population-based validation. We hope to find out the underlying biomarkers for diagnosis and prognosis, and provide more opportunities to understand the degenerative cervical diseases.

骨赘形成是颈椎病引起神经、血管压迫的常见原因，深入研究其成骨现象对揭示该病的病理机制具有重要意义。近年研究表明，椎间盘纤维环成纤维细胞具有成骨化生潜能，是形成骨赘的潜在细胞来源，而环形RNA是一类新的非编码RNA，可调控基因表达，已证实与神经退变性疾病等相关，课题组前期研究表明，椎间盘纤维环成纤维细胞具有特异的环形RNA表达谱，其部分与细胞增殖、转化等相关，因此，我们认为特定环形RNA的表达异常可能参与了其成骨化生过程。本研究拟深入筛选分析退变性颈椎间盘纤维环成纤维细胞中环形RNA的表达特点，研究环形RNA在骨赘形成中的作用及其机制；此外，环形RNA结构相对稳定，可在血清中检测，课题组拟进一步比较分析环形RNA血清分布特点，筛选敏感特异的环形RNA，并在成骨诱导模型及不同人群中验证，力图寻找可以预测骨赘形成趋势的诊断标志物，为深入探究颈椎病的病理机制和指导临床实践提供新的视角。

随着人口老龄化程度加深和现代生活方式的转变，颈椎病发病率逐年增高，其作为常见病之一，严重影响人民群众健康。颈椎病发病重要病理机制之一是骨赘形成，进而压迫血管、神经等结构引起相应临床症状。颈椎间盘成纤维细胞具有成骨化生潜能，可能与骨赘形成相关。课题组前期实验发现环形RNA在颈椎间盘纤维环成纤维细胞中具有特异性表达，进而在本课题中深入研究该类环形RNA在成骨化生过程中的作用机制。课题组在研究中成功建立颈椎间盘纤维环原代细胞分离技术体系，并在其中发现了一类具有间充质干细胞样特性的细胞群，该类细胞群具有特异性细胞表面标记物，即CD90-FITC、CD105 PerCP-cy5.5、CD73-APC阳性，CD45、CD34、CD11b、CD19、HLA-DR-PE 阴性的细胞群，能够自我复制更新，并能向成骨、成软骨、成脂肪三个方向定向分化，项目组通过流式细胞分选技术进行细胞筛选，建立了细胞库，并构建了该类细胞成骨化生的细胞分化模型。利用高通量测序技术，课题组研究发现282个环形 RNA 与纤维环组织干细胞的成骨分化过程特异性相关；对环形RNA与所在HOST基因表达特点进行了分析，归纳出可能存在九种相互作用关系模式；采用GO分析从中进一步筛选了包括11个特异性高表达候选环形RNA便于进行后续的深入研究。同时，课题组还在颈椎病合并骨质疏松症患者血浆外泌体中检测到环形RNA特异性表达，采用高通量测序技术筛选分析，进一步通过队列研究初步验证，circZCCHC7存在miR-6790-3p，miR-1236-3p，miR-6821-3pmiRNA结合位点，可能与其结合参与与骨代谢过程，可作为潜在候选诊断标志物。