Islet-1与Akt竞争破解MSCs干性促其心肌细胞特化机制研究

As the seed cells of transplant treatment for heart disease, researches of MSCs obtained significant breakthrough in both basic and clinical. But recently, the mechanism of MSCs differentiation into cardiomyocyte was not clear. And clinical application of MSCs was restricted because of low efficiency and extent of differentiation. In years of related researches we found that there exist different temporal orders of cardiac-specific genes expression in different methods of inducing MSCs differentiation into cardiomyocyte. We speculate that the reason was the stemness of MSCs. Combined with previous research and related literature, we proposed the hypotheses: Akt and Islet-1 competitive binding to the differentiation epigenetic recruit complexes, and the competitive relationship is the key for MSCs deciding to stemness maintain or differentiation, and regulate both Akt and Islet-1 could break stemness of MSCs and induce MSCs differentiation into cardiomyocyte more efficient. By parsing Akt activity interactively regulated by epigenetic modification and break the stemness maintenance mechanism in the process of Islet-1 inducing MSCs differentiation into cardiomyocyte-like cells, the research intends to lay the scientific foundation of experiments for targeted intervention of MSCs pecific differentiation into cardiomyocytes, improve the differentiation efficiency and extent of differentiation , and ultimate realize the clinical application of MSCs, highlight the need to address the MSCs cardiomyocyte specific differentiation.

MSCs作为移植治疗心肌损伤疾病种子细胞之一，无论基础还是临床应用研究均有较大突破。但目前对MSCs向心肌细胞分化的机制尚未完全明了，且分化率和程度不高，使临床应用受限。多年相关研究我们发现，不同方法诱导MSCs向心肌细胞分化，心肌特异基因表达存在时序差异，究其原因推测是MSCs对自身干性的维持所致。结合前期研究及相关文献，我们提出科学假设：在MSCs分化表观调控招募蛋白复合体中，Akt与Islet-1竞争结合状态是其保持干性或进入分化的关键；调控Akt与Islet-1能够打破MSCs干性、更好地促进向心肌细胞分化。本研究拟通过解析在Islet-1诱导MSCs向心肌样细胞分化过程中，表观遗传修饰交互调控Akt的活性、并通过竞争结合破解干性维持机制，为MSCs向心肌细胞特异性分化靶点干预、提高分化效率和分化程度，最终实现MSCs临床应用奠定科学的实验基础，同时也为该类研究提供更好的探索途径。

该研究旨在探明MSCs心肌诱导过程中，其干性与分化的平衡关系调控机制，以及找到关键因子进行平衡关系调控，促进心肌分化。根据原实验计划，发现Akt与干性因子的变化趋势没有显著的内在联系，并且Akt活性对Islet-1与GCN5的结合的影响可能与间充质干细胞的干性变化无直接联系。因此课题组进行了思路调整，发现调控干性的β-catenin同时也与心肌分化有密切联系，于是我们建立了敲低β-catenin的C3H10T1/2细胞模型，进一步发现敲低β-catenin7天后，细胞出现显著的心肌样细胞分化。通过CO-IP，CHIP，荧光素酶报告系统，同位素示踪等实验发现，MLIP与GCN5竞争结合 Islet-1，共同参与C3H10T1/2细胞向心肌分化过程。敲低β-catenin会显著上调E3泛素化连接酶UBE3C的表达，从而修饰MLIP泛素化导致其表达降低。同时，会通过抑制E3泛素化连接酶WWP1的表达上调Islet-1，进一步促进Islet-1与GCN5的结合，从而促进间充质干细胞向心肌细胞分化。我们的实验结果说明了β-catenin在诱导间充质干细胞向心肌分化过程中具有关键作用，建议在分化过程中可以作为一个潜在的关键因子加入到分化策略中。