基于单细胞组学的椎间盘髓核细胞亚群分析及其在退变中的作用与机制研究

Disc degeneration associated diseases is an important disease that greatly affects the quality of life in modern society. However, the lack of precisive approaches to effectively hinder or reverse the degeneration process is largely due to poor understanding of the pathophysiology of both the disease and the disc itself. Recent findings showed that heterogeneity exists in intervertebral disc cells, especially in nucleus pulposus cells, which means there are many cell subtypes in the nucleus pulposus. Thus unveiling the functional role of these cell subtypes in degeneration may be the key to decipher the mechanism of intervertebral disc degeneration. However, related studies are still at the preliminary stage, and due to the limited number of tissue cells, lack of research methods and experimental design issues, the data demonstrated varies greatly between studies. Taking advantage of single-cell sequencing technology, our group, for the first time, performed single-cell high-throughput sequencing analysis on normal disc nucleus pulposus cells. By bioinformatics analysis of the data, three different cell subtypes with representative markers were found in normal disc nucleus pulposus cells. On such basis, our team aimed to analyze the differences of degenerative nucleus pulposus cells by single-cell sequencing by comparing the differences between degenerated and non-degenerated nucleus pulposus cells, thus revealing the potent pathogenic cell subpopulation and key genes in intervertebral disc degeneration. And by using both in vitro and in vivo experiments to verify the mechanism. Through this project, we hope to delineate the mechanism of disc degeneration and provide theoretical foundation and new ideas to accurate diagnosis and treatment.

椎间盘退变相关疾病是影响现代社会人们生活质量的重要疾病之一，迄今仍缺乏延缓或逆转其病变进程的有效早期精准治疗靶点与方法，而这与其发病机理不明确密切相关。近来研究表明，椎间盘髓核细胞存在异质性，即髓核组织存在多种细胞亚群，因此找到并明确髓核细胞持续退变的关键细胞亚群可能成为突破椎间盘退变研究瓶颈的关键点。然而，目前国内外的相关研究均处于初步探索阶段，结果差异大、重复性差。本课题组基于单细胞测序技术，前期利用大量临床样本，首次针对正常椎间盘髓核细胞进行单细胞高通量测序分析，并通过生物信息学分析已发现3组具有代表性标志物的髓核细胞亚群。在此基础上，本次研究拟针对椎间盘髓核细胞退变前后的差异，应用单细胞测序进一步分析比对，找到真正致病的潜在细胞亚群及关键基因，并在体外及体内实验中验证其机制。本研究将揭示椎间盘退变机制的新途径，为早期精准诊疗奠定理论基础。

椎间盘退变相关疾病是影响现代社会人们生活质量的重要疾病，迄今缺乏逆转其病变进程的有效早期精准治疗方法，这与其发病机理仍不明确有关。近来研究表明，椎间盘细胞存在异质性，即髓核组织存在多种细胞亚群，因此找到并明确髓核细胞持续退变的关键细胞亚群可能成为突破椎间盘退变研究瓶颈的关键点。然而，目前国内外的相关研究均处于初步探索阶段，结果差异大、重复性差。本课题组通过两种不同单细胞转录组测序技术的比较，找寻出具有优势的测序方式，完成全部有效单细胞测序总数达4万例，并针对该部分数据进行生物信息学分析，明确其致病网络及细胞亚群。通过针对关键基因构建基因敲除模型，并通过体内及体外实验明确了致病关键基因CHSY、CHST以及CHI3L1在椎间盘退变过程中的作用与机制，发现了具有潜在靶向治疗价值的关键靶点。除此之外，在数据挖掘分析过程中，发现性别差异基因ORM具有退变相关性，并针对性别相关ORM蛋白进行了系统的体内体外研究，明确了ORM1的表达具有性别相关性与退变相关性，构建了ORM1的基因敲除模型小鼠并验证了ORM1具有调控髓核细胞退变作用与机制。相关研究内容以论著形式发表相关SCI期刊论著13篇，其中影响因子10分以上2篇，培养硕士研究生2名。相关成果获上海市医学科技奖一等奖，申请国家发明专利2项。