脂肪间充质干细胞向限定性内胚层细胞重编程过程中长链非编码RNA调控作用的研究

The strategy of generation transplantable insulin-secreting cells holds great promising for diabetes treatment. Our previous studies proved that adipose tissues derived mesenchymal stem cells (AD-MSCs) would be reprogrammed towards definitive endoderm (DE) cells, and subsequently differentiation into pancreatic progenitor cells by mimicking pancreatic development signaling pathway. However, those studies mainly focused on selection of seed cells, optimization of induction medium and detection of specific marker, and so on. Superimposed on this classical genetic program, epigenetic mechanisms, which also control the fate of stem cells, remain largely unknown. Recently, it has been found that a significant proportion of lncRNAs (long noncoding RNA) are physically associated with chromatin-modifying complexes such as EZH2. Those lncRNAs could guide chromatin-modifying complexes to specific genomic loci to regulate gene expression, and affect differentiation. In this project, we choose the crucial stage of differentiation of AD-MSCs into pancreatic progenitor cells, i.e. reprogramming of AD-MSCs to DE as research model, to detect the changes of histone methylation of the key transcription factors during this progress. Moreover, we could use an approach based on in vivo biotinylation system in combination with RNA immunoprecipitation (Bio RIP) to identify functional lncRNAs which would be associated with EZH2, and explore the regulatory mechanisms underlying.

获得临床移植用胰岛素分泌细胞是糖尿病治疗研究的新方向。既往的研究多围绕着种子细胞的选择、诱导体系的优化和关键标志物的检测等开展，而很少涉及分化的分子机制，尤其深入到表观遗传学水平的研究更为缺乏。我们前期的研究证实成体脂肪组织来源的间充质干细胞（AD-MSCs）体外经过诱导可以重编程为限定性内胚层（DE）细胞，进一步可诱导为胰岛素分泌细胞。最近的研究表明一些长链非编码RNA(lncRNA)可以结合组蛋白甲基化酶EZH2所在的复合物，指导其对靶基因组蛋白甲基化状态进行修饰，进而影响干细胞的命运决定。因此在本项目中，我们选择诱导的关键步骤即AD-MSCs向DE细胞重编程过程作为模型，研究关键转录因子的组蛋白甲基化修饰状态的变化，并采用体内生物素技术结合RNA免疫共沉淀（Bio RIP）的方法，研究与EZH2结合的lncRNA在其中所发挥的作用，并对其调控机制进行深入探讨。

脂肪间充质干细胞是一种理想的种子细胞，它可以向限定性内胚层细胞诱导分化，为获得具有正常功能的肝脏和胰腺细胞提供有价值的选择。本项目旨在探寻MSCs向DE分化过程中关键转录因子组蛋白修饰水平的变化，以及其中发挥关键作用的非编码RNA并探索其作用机制。首先我们优化了已有的诱导分化体系，进一步提高了MSCs向DE分化的效率，其次我们证明诱导成功的DE细胞具备向肝脏和胰腺细胞的分化能力，并用动物模型在体内验证了得到肝细胞的获得。接着我们采用CHIP-seq的方法对MSCs向DE分化过程中关键转录因子的组蛋白甲基化修饰水平进行了分析。同时我们还采用了基因表达谱、单细胞测序技术对MSCs向DE，以及后续的向肝细胞分化过程的基因表达变化进行了解析。最后我们对在分化过程中起重要作用的一条非编码RNA，并对起作用机制进行了探讨。通过本项目的研究，我们更提高MSCs向DE分化的效率，更深入的了解了分化的分子机制，为进一步解析MSCs分化和命运决定的提供了重要参考。.利用本项目积累的经验和技术，项目组成员还完成关于长链非编码lncRNA在MSCs诱导乳腺癌细胞EMT过程中发挥的作用和机制的一项研究工作。项目执行期间还资助了项目组成员在不同因素（肿瘤分泌的exsome和低强度激光）处理MSCs后对MSCs表型、分化能力和迁移等生物学特性影响的研究。这些研究加深了我们对MSCs在不同环境变化中应答的理解。