炎症微环境通过YTHDF2调控KAISO m6A甲基化介导强直性脊柱炎骨化进程的机制研究

Ankylosing spondylitis (AS) is an autoimmune disease characterized by inflammation and ossification. The inflammatory microenvironment may be an important factor in the ossification of AS, but the intermediate mechanism is still not fully understood. Our previous studies observed that the levels of Kaiso protein antibody are elevated in early stage of AS but decreased in the late stage. In vitro experiments, we confirmed that Kaiso protein can inhibit osteogenic differentiation of ligament MSCs and the expression of Kaiso protein is regulated by methylation reader protein YTHDF2. Moreover the expression of YTHDF2 is affected by the inflammatory microenvironment. These results suggested that inflammation may act on YTHDF2 to regulate the methylation level of Kaiso and thus affect osteogenic differentiation. Focusing on this hypothesis, this topic will be carried out: ①To study the role of Kaiso in the osteogenesis of ligament MSCs induced by inflammation; ②To investigate whether YTHDF2 affects the osteogenesis of ligament MSCs by regulating the level of Kaiso RNA methylation; ③Detection of YTHDF2 expression and Kaiso methylation levels in clinical samples and animal models, then analysis their correlation with inflammation and ossification. This research can reveal the possible mechanism of inflammation to ossification in AS, and provide new ideas for the study of epigenetics in the diagnosis and treatment of AS.

强直性脊柱炎（AS）是以炎症和骨化为主要特征的自身免疫性疾病。炎症微环境可能是导致AS骨化的重要因素，但具体机制尚不清楚。我们前期研究提示Kaiso蛋白抗体在AS早期炎症活动阶段表达升高，而晚期相对降低；体外实验证实Kaiso蛋白可抑制韧带MSCs成骨分化且Kaiso表达受到甲基化识别蛋白YTHDF2的调控，而炎症微环境可影响YTHDF2表达。这提示炎症可能作用于YTHDF2来调控Kaiso甲基化水平进而影响成骨分化。围绕该假设，本课题将展开：①Kaiso分子对炎症微环境诱导韧带MSCs成骨分化进程的调控；②YTHDF2是否通过调控Kaiso分子RNA甲基化修饰水平进而影响韧带MSCs成骨分化进程；③临床样本及动物模型中检测YTHDF2表达和Kaiso甲基化水平并分析与炎症和骨化的相关性。本课题可初步揭示AS由炎症转为骨化的可能机制，并为表观遗传学在AS诊断及治疗中的研究提供新思路。

背景：强直性脊柱炎（ankylosing spondylitis,AS）是以炎症和骨化为主要特征的自身免疫性疾病。炎症微环境可能是导致AS骨化的重要因素，相关研究显示甲基化修饰在成骨进程中有重要作用，但甲基化修饰在AS炎症及骨化进程中的作用及相关机制仍需探索。.研究内容：AS甲基化水平及相关作用酶的筛选及验证；强直性脊柱炎血清Kaiso自身抗体的验证及相关性分析、Kaiso蛋白功能的探索；甲基化酶YTHDF2是否通过调控Kaiso分子RNA甲基化修饰水平影响AS韧带间充质干细胞成骨分化进程。.重要结果：AS组织样本验证晚期AS患者总甲基化水平及YTHDF2表达升高，体外实验结果显示高浓度炎症微环境抑制YTHDF2介导的韧带间充质干细胞甲基化水平，而低浓度炎症微环境增强该进程；细胞实验结果显示AS早期高表达的Kaiso蛋白抑制骨化进程，而晚期低表达的Kaiso促进骨化进程；进一步机制探究显示炎症可能作用于YTHDF2来调控Kaiso甲基化水平进而影响成骨分化。.科学意义：基于本课题的研究，我们初步揭示甲基化修饰在AS由早期炎症转为晚期骨化中的可能机制，为AS炎症-骨化进程的转变提供新的认识，并为表观遗传学在AS诊断及治疗中的研究提供新思路。