LncRNA H19经外泌体运载调控软骨细胞自噬防治骨关节炎的机制研究

Osteoarthritis (OA) is a degenerative joint disease. There is a continuing need for safe and effective therapies. Recent research has focused on the mechanisms that maintain cell and tissue homeostasis. Autophagy is a cellular homeostasis mechanism for the removal of dysfunctional organelles and macromolecules; and also modulates OA-related gene expression in human chondrocytes. Our primary studies about OA chondrocytes have revealed that long non-coding RNA (LncRNA) is an important class of genes involved in various biological functions. Through bioinformatics data analysis we hypothesize that LncRNA H19 is potentially the most significant connection with OA. The aim of this study is to apply LncRNA H19 carried by bioengineered exosomes for regulating cartilage autophages and homeostasis. Through a series of in vitro cell experiments and in vivo animal experiments, we expect to assess the cartilage autophage related molecular mechanism of OA, to understand the biological processes of LncRNA H19 in cartilage regeneration, to provide evidences and therapeutic paradigm for prevention and treatment of OA using LncRNA H19 carried by bioengineered exosomes, and to improve the quality and efficiency of cartilage regeneration.

骨关节炎（OA）严重危害人类健康，目前其机制研究主要集中于软骨细胞和组织的稳态平衡，其中自噬发挥着重要的作用。且长链非编码RNA（LncRNA）与OA疾病进展及自噬均联系紧密。经生物信息学分析，我们推测LncRNA H19可能与OA的发病紧密相关，并初步通过临床样本检测证实LncRNA H19与自噬在OA中呈现早期代偿性增加，晚期失代偿减低的规律。前期研究发现LncRNA H19可经外泌体搭载及富集，并传递给靶细胞，调控靶细胞的生物学功能。本课题拟在前人和我们前期研究基础上，利用生物工程化外泌体搭载LncRNA H19来调控软骨细胞自噬以维持软骨稳态平衡，利用体外细胞学、体内动物学和分子生物学实验，通过检测软骨和自噬相关指标进行验证，初步阐明LncRNA H19经调控OA软骨细胞自噬促进软骨再生的途径和分子机制，为生物工程化外泌体运载LncRNA H19高效防治OA提供治疗策略和科学依据。

骨关节炎是一种常见的退行性骨关节疾病，目前缺少有效的预防或治疗手段。课题组在前期建立了稳定的间充质干细胞分离培养体系，并在此基础上成功建立了在外泌体中稳定高表达目标分子的运载体系，如运载LncRNA H19（H19-OL-sEVs）、circRNA3503（circRNA3503-OE-sEVs）和ZEB1（fC-sEVs-ZEB1）。为了在体内充分发挥外泌体的作用，课题组应用了三种递送技术（海藻酸钠水凝胶、温敏水凝胶PDLLA-PEG-PDLLA、自组装聚合物涂层HA/PLL），并通过一系列实验证实三种递送技术均能有效负载外泌体，延长其在体内作用的时间，取得更好的效果。体外实验证实这些方法均能有效增强细胞的增殖、迁移能力，抑制细胞凋亡，能够促进组织再生相关蛋白的表达，减少组织损伤相关蛋白的表达。通过常规组织学染色、免疫荧光和免疫组化等一系列体内实验证实它们的组织再生和组织保护作用。本项目探索了外泌体高效运载和表面功能化等技术，为维持关节软骨微环境稳态，延缓骨关节炎进展提供了新思路。