POTEB抑制Notch通路在软骨终板干细胞向髓核细胞分化中的作用机制研究

The intervertebral disc tissue engineering now becomes the popular research for The threapy of degenerative disc diseases.It is the first time for us to demonstrate the presence of stem cells in the human degenerated intervertebral disk cartilage endplate，which named CESCs（stem cells derived from the cartilage endplate）.CESCS cocultured with NP cells were successfully differentiated into NP-like cells.We suggested that CESCs may be a new source of seed cells for managing IVD degeneration in the future.However, the underlying molecular mechanisms of the CESCs differentiation are still unclear.Our previous studies have demonstrated that CESCs were successfully differentiated into NP-like cells through exosome-mediated by NP.MS found that POTEB existed in exosome.Also we found POTEB is highly expressed in nucleus pulposus cell than chondrocyte.As cocultured,Those NP whose poteb is inhibited express low capacity of differentiation.It is indicated that POTEB plays a great important in CESCS differentiation. Notch may induce Twist1 to inhibit chondrogenic differentiation via direct Notch-NICD/RBPJK binding to its promoter region.POTEB's domain is highly similar with the Notch intracellular domain（NICD）. we infer that POTEB can inhibit Notch pathway by binding to CSL and RBPJk and increase the capacity of differentiation of cartilage endplate stem cell to nucleus pulposus cell. We construct the Twist1 promoter-driven luciferase reporter and choose chromatin immunoprecipitation in order to clarify how POTEB modulates the differentiation of CESCs.

组织工程髓核为治疗椎间盘退行性疾病提供可能。课题组率先报道了软骨终板干细胞(CESCs)，其与NPC共培养可诱导分化为NPC，有望成为新的种子细胞来源。但其诱导分化的分子机制尚不清楚。前期研究发现NPC通过外分泌体传递信号诱导CESCs分化。质谱发现外分泌体中存在POTEB蛋白，POTEB是我们前期发现的NPC区别于软骨细胞的高表达蛋白，抑制POTEB表达的NPC诱导CESCs分化为NPC的能力明显降低。提示POTEB在CESCs向NPC定向分化中具有重要的作用。Notch中NICD1与核内CSL-RBPJκ形成复合体调控Twist1启动子活性抑制软骨形成，POTEB序列与NICD结构域高度相似，推测POTEB与Notch竞争结合抑制Notch通路，促进CESCs向NPC分化。为此我们拟采用荧光素酶报告基因、染色质免疫沉淀等方法以阐明POTEB在CESCs向NPC定向分化中的作用及其机制。

一、项目背景.下腰痛的病理基础为髓核细胞数量减少和功能降低。为治疗该疾病，干细胞疗法被认为是最有前景的方法。.本课题组前期通过基因芯片发现髓核细胞（NPC）中POTEB的可作为其标志物。进一步研究发现其可促进软骨终板干细胞（CESC）向NPC分化。.同时，外泌体的研究发现其在多种生物学效应中有重要作用。我们前期发现NPC外存在大量分泌性囊泡，其内含有POTEB。但其是否通过外泌体发挥作用，且通过何种方式影响了该生物学过程，为本课题研究的目标。.二、主要研究结果.1. 髓核细胞外泌体的提取与鉴定.通过磷钨酸染色发现，NPC-外泌体与经典的外泌体大小形态一致，其表达外泌体标志蛋白CD63、Tsg101，无Calnexin表达。.2.CESCs对NPC-外泌体的摄取能力检测.利用PKH67对NPC-外泌体进行标记，结果显示，CESCs中可观察到点状绿色荧光，而PKH67对细胞染色呈现出对细胞膜的染色。.3.NPC-外泌体对CESCs的诱导分化作用.用NPC-外泌体对CESC进行刺激3、7、10、14天后，发现各时间点CESC都向NPC分化，且存在时间依赖效应。.4.NPC-exosomes与间接共培养方式对诱导CESCs分化能力的比较.分别采用NPC-外泌体与间接共培养方式刺激CESCs14天后发现，外泌体组的分化效应明显强于间接共培养组。.5.在CESC向NPC分化过程中，POTEB可以抑制Notch通路中的各关键蛋白。.在CESC向NPC分化过程中，Hes1，5，7以及Hey1，2的表达均降低。其中Hey2的表达下降最为明显。并且，Hey2的表达在7天之后会持续下调。.6.抑制NICD3可以促进CESC向NPC分化。.在CESC向NPC分化过程中，抑制NICD3其分化作用最强，但是在抑制NICD1，2，4中，对其分化能力影响较弱。.7.POTEB在CESC向NPC定向分化过程中可向核内迁移，并可与RBP-jκ相结合.用NPC外泌体诱导CESC向NPC分化7天后，我们发现POTEB可穿过细胞核膜向核内转移。免疫共沉淀显示POTEB可以与RBP-jκ相结合。.三、科学意义. 本研究发现NPC来源的的外泌体可通过经典方式与CESC结合并刺激其向NPC分化。并且，POTEB可通过定向向细胞核内转移，与RBP-jκ分子结合抑制Noth信号通路，从而促进CESC向NPC分化