LncRNA-32598对Sox9转录和转录后调控机制在干细胞来源软骨细胞表型维持中的作用研究

The phenotype maintenance of seed cells in tissue engineered cartilage is the current focus. Sox9 is a key factor that regulates the phenotype of chondrocytes. LncRNA plays an important role in the chondrocyte differentiation. But its mechanism of regulation of Sox9 is unknown. In preliminary experiment，we had confirmed that miR-145 targeting Sox9. Through high-throughput sequencing, we found that Lnc-32598 was significantly up-regulated during chondrocyte differentiation and Lnc-32598 competed with Sox9 for binding to miR-145. After interfering the LncRNA expression, the Sox9 expression was reduced on both protein and mRNA level, which was difficult to be explained only by miR-145-Sox9 post-transcriptional regulation. These results suggest that there are other regulation manners for Sox9 on the transcriptional levels. Mass spectrometry evidence showed that the LncRNA was combined with FXR1 which recruited STAT3 to bind to the Sox9 enhancer, proposing Lnc-32598/FXR1/STAT3 regulation of Sox9 was involved in chondrocyte differentiation. This study tries to find out the potential ceRNA expression pattern of Lnc-32598 on the regulation of Sox9 by in vivo and in vitro experiments, and the effect of LncRNA pathway on cartilage repair will be confirmed by animal model. This study will provide theoretical guidance for cartilage repair performed by tissue engineering.

组织工程软骨种子细胞表型保持是目前的重要方向，Sox9为调控软骨细胞表型的核心因子。LncRNA在软骨分化中发挥重要作用，但其对Sox9的调控机制不详。在前期证实miR-145靶向调控Sox9基础上,高通量测序发现Lnc-32598在软骨分化中显著上调，且与Sox9竞争性结合miR-145；干扰该Lnc则Sox9蛋白和核酸均降低，这难以通过miR-145-Sox9单一转录后调控阐释，推测还存在针对Sox9转录水平的调控机制。质谱证据发现该Lnc可与FXR1结合，后者可募集STAT3结合到Sox9增强子，故提出Lnc-32598/FXR1/STAT3调控Sox9参与软骨分化假说。拟以Sox9的CeRNA调控网络为基础，通过体内外实验证实上述相关分子的表达规律，阐明Lnc对Sox9两种调控细节，并利用动物模型明确干预该Lnc路径对软骨修复的影响。本研究将为组织工程策略进行软骨修复提供理论指导。

通过构建组织工程软骨进行关节软骨损伤修复是目前的研究热点，但在组织工程软骨构建过程中易出现软骨的早期退变，同样骨关节炎病理过程也主要呈现关节软骨基质的退行性病变。软骨细胞作为软骨组织中的唯一细胞类型，其功能正常与否是维持细胞外基质稳定的关键因素。Sox9为调控软骨细胞表型的关键转录因子，探求软骨分化以及软骨退变中对Sox9的调控机制，有助于为防止组织工程软骨退变，稳定种子细胞表型提供新的研究思路。LncRNA作为表观遗传调控的重要方式之一，在骨骼系统中日益受到重视。LncRNA的作用机理为我们提供了一种全新的视角，用来进行阐释其调控Sox9的调节机制，尤其是在软骨细胞表型调控的分子机制方面，有望为软骨退变的防治获得一些新的干预策略依据。 .课题组建立了原代间充质干细胞体外软骨分化以及肥大化分化两种诱导模型，检测了其lncRNA及miRNA的表达谱。利用小鼠来源C3H10T1/2细胞，建立了稳定的体外诱导分化模型；通过Real-time PCR技术，对C3H10T1/2细胞不同分化阶段LncRNA-32598进行干预。证实LncRNA-32598在间充质干细胞成软骨以及肥大化分化两个阶段中均发挥重要作用，即LncRNA-32598可促进软骨细胞分化，并且抑制软骨细胞的肥大化分化。机制上，课题组发现存在LncRNA-32598/miR-145（胞内）以及LncRNA-32598/FXR1/STAT3（核内）两种调控Sox9表达参与软骨细胞分化的机制。通过干预LncRNA-32598及其下游信号通路，可有效调控软骨细胞的表型稳态保持及下调退变相关基因表达。利用小鼠骨关节炎动物模型，课题组发现lncRNA-32598能够有效维持关节软骨的正常结构和代谢稳态，过表达LncRNA-32598可延缓骨关节炎进展。.另外本项目以软骨细胞表型调控为出发点，建立了软骨内成骨修复体系，探讨了LncRNA和Ce纳米颗粒等因素对软骨表型的影响及其在软骨内成骨体系中的作用。该研究从LncRNA角度初步探讨了软骨细胞表型调控的机制及其在软骨损伤疾病中的病理生理作用，结果将为临床上软骨损伤、骨关节炎防治提供新的治疗靶点。本项目获授权中国发明专利1项，日本发明专利1项；发表课题相关SCI论文7篇，参加国内外学术会议10次；指导毕业博士生1名，毕业硕士生2名。

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