Lnc-NP调控ADSC成髓核中晚期分化中FoxA2/Shh信号通路的机制研究

Based on our group and colleagues’ research work, the harsh microenvironment obviously constrained the trans-differentiation of stem cells into nucleus pulposus cells, which is the bottleneck of the biotherapy treatment for disc degenerative disease. The trans-differentiation of human adipose stem cells (hADSC) into nucleus pulposus cells is strictly scheduled, and mid-advanced stage mainly is regulated by the Forkhead box protein A1/2 (FoxA1/2) and sonic hedgehog (Shh) signaling pathways. LncRNA is one of the important regulatory factors for the trans-differentiation of stem cells. We previously found that both Lnc-NP and FoxA2 were highly expressed in middle-late trans-differentiation of hADSC into nucleus pulposus, Lnc-NP located in hADSC cytoplasm, and FoxA2 was binding protein of Lnc-NP. The trans-differentiation into the nucleus pulposus was stagnation in the early period after Lnc-NP is RNAi interferenced, and the expression of nucleus pulposus antigen and FoxA2 decline significantly. We hypothesized that hADSC into nucleus pulposus mid-advanced established mainly by Lnc, NP, FoxA2 and Shh regulation. So the established in vitro to clarify Lnc-NP, cross talks between the FoxA2 and Shh, established a complete set of RNA research system, and use gene transfection to have top 1ncRNA - hADSC nucleus pulposus differentiation; Validation of heterogeneous allografts as the nucleus pulposus differentiation after 1ncRNA-hADSC. The specific molecular mechanisms of 1ncRNA will be clarifed in trans-differentiation of hADSC into nucleus pulposus cells, which provides experimental data and new targets for stem cell transplantation in the treatment of disc degeneration.

干细胞成髓核分化受限是退变椎间盘治疗的瓶颈。人脂肪干细胞(hADSC)成髓核分化具有时序性，而中晚期阶段主要受FoxA1/2和Shh调控。lncRNA是干细胞分化的重要调控因素。我们前期发现Lnc-NP和FoxA2同时高表达于成髓核中晚期分化的hADSC，Lnc-NP定位于胞浆，FoxA2是其结合蛋白；且Lnc-NP被RNAi干扰后成髓核分化将停滞在早期阶段，髓核抗原和FoxA2表达也显著下降。我们推测hADSC成髓核中晚期分化主要受Lnc-NP、FoxA2和Shh调控。故拟在体外阐明Lnc-NP、FoxA2和Shh间的交叉对话，建立一整套RNA研究体系，并借助基因转染获得高成髓核分化的1ncRNA-hADSC；进而验证异种异体移植成髓核分化后的1ncRNA-hADSC修复大鼠退变椎间盘的效果；阐明1ncRNA在hADSC成髓核中晚期分化中的作用，从而为椎间盘退变的治疗提供新数据及新靶点。

基于脂肪组织的干细胞（ADSC）疗法有望治疗椎间盘（IVD）变性，但诱导髓核（NP）样分化的困难限制了其临床应用。越来越多的研究证明，长链非编码RNA（LncRNA）在干细胞定向分化中起到了很重要的作用。我们期望以RNA测序为基础，发现调控ADSC成髓核细胞分化的关键LncRNA，并研究其与相关信号通路、转录因子之间的调控机制，进而调控关键靶点，提高ADSC成髓核细胞分化的效率，以期后续能够应用于临床。研究内容、结果及数据主要包括：.1）分离、纯化、培养和鉴定人脂肪干细胞（hADSCs），证实了hADSCs的成骨、成脂及成软骨多向分化潜能；.2）采用3D细胞微球的方法，在模拟椎间盘微环境情况下，采用改良成髓核诱导培养基培养hADSCs（髓核组）、改良成软骨诱导培养基培养hADSCs（软骨组），正常培养基培养hADSCs（正常组）；.3）利用RNA-seq测序技术获得了髓核组、软骨组、正常组LncRNAs和mRNA的差异表达谱；.4）利用已有数据库及生信分析等筛选出了hADSCs成髓核分化的候选LncRNAs 25个；.5）应用RT-PCR对筛选出的候选LncRNAs进行验证，并最终筛选出差异最明显的LncRNA，命名为LncNP；.6）利用慢病毒将LncNP转染到hADSCs中，获得了过表达LncNP的成髓核定向分化的干细胞LncNP-hADSCs；.7）探索LncNP调控hADSCs成髓核分化的机制，同时发现FoxA2可通过shh通路促进成髓核分化；.8）将预髓核分化的LncNP-hADSCs注射入SD大鼠的退变椎间盘内，发现其具有修复退变椎间盘的效果；.9）发表SCI论文共计12篇，其中LncRNA相关综述2篇，最高影响因子8.063分，5分以上5篇，部分成果参加了中国骨科相关的会议；.10）培养博士生3名，硕士生2名；.本项目总结了LncRNA在软骨发育、病变、再生方面以及干细胞分化领域的重要作用，并发表了两篇相关的SCI文章，同时通过测序发现了促进hADSCs分化成髓核细胞的LncRNAs，命名为lncNP，利用慢病毒构建了过表达lncNP的hADSCs，其具有优良的成髓核细胞分化的能力，在体内研究中也展现出了修复退变椎间盘的效果，为未来干细胞治疗椎间盘退变的临床医学转化研究奠定实时监控和个性化治疗的理论基础，对临床治疗具有一定指导意义。