脯氨酸羟化酶小分子抑制剂FG-4497诱导间充质干细胞动员的机理研究

Mobilization of mesenchymal stem cells (MSCs) is a promising strategy for tissue repair and regenerative medicine, which may avoid the problems of administering cultured expanded MSCs in vitro. There is little evidence for reproducible, effective methods for mobilizing MSCs. The mechanisms responsible for MSC mobilization are currently unclear. In our previous study, we revealed, for the first time, that HIF-1α plays a pivotal role in controlling hypoxia-induced MSC mobilization (Stem Cells and Development 2011 20(11):1961-71). Prolyl hydroxylase domain protein (PHD) is the rate-limiting enzyme controlling the degradation of HIF-1. HIF prolyl hydroxylase inhibitors (PHI) inhibit the degration of HIF-1α and can stabilize its protein expression. Recently, we found that mouse MSCs can be mobilized into peripheral blood by novel small molecular PHI FG-4497. Emerging evidence indicated that HIF-1 reguated SDF-1α/CXCR4 and VEGF/VEGFR pathways may play an important role in stem cell mobilization. These downstream signals may regulate the mobilization process through effect on bone marrow environment and MSCs themselves. Based on our previous data and aboundent other evidence, we will firstly confirm the MSC mobilization effects of FG-4497 in this study, and determine the characteristics of MSCs derived from mobilized peripheral blood. Secondly, we will clarify the mechanisms of mobilization of FG-4497 and investigate the role of HIF-1 and its downsteam signals by virous methods, with particular emphasis on the role of SDF-1α/CXCR4 and VEGF/VEGFR pathways. Our study will provide a novel insight into the mechanisms responsible for MSC mobilization and may help in the development of clinically useful therapeutic mobilizing agents.

间充质干细胞（MSCs）动员方法的建立可以避免体外培养后移植治疗的缺点，提供低创有效的新途径。目前临床上尚无有效的MSCs动员方法，动员机制尚未明确。在前期研究中，我们首创性地证实缺氧诱导因子-1（HIF-1) 是缺氧诱导MSCs动员的关键因子（Stem Cells Dev.）。脯氨酸羟化酶是调控HIF-1降解反应的限速酶，脯氨酸羟化酶抑制剂（PHI）可以稳定HIF-1的表达。我们预实验发现，小分子PHI FG-4497可以动员小鼠MSCs。研究提示：HIF-1介导的SDF-1α/CXCR4通路与VEGF/VEGFR通路可能通过作用于微环境与MSCs本身而调控MSCs动员。本项目拟研究：（1）FG-4497对MSCs的动员作用与动员外周血来源MSCs生物学特性；（2）HIF-1及下游信号通路在介导FG-4497动员MSCs中的作用机理。本项目的研究将为MSCs动员剂的开发提供重要实验依据。

间充质干细胞（MSCs）动员方法的建立可以避免体外培养后输注治疗的缺点，提供低创有效的新途径。目前尚无临床可行的MSCs动员方法，动员机制尚未明确。本项目组前期研究首创性地证实了缺氧诱导因子-1（HIF-1）是缺氧诱MSCs动员的关键因子。脯氨酸羟化酶是调控HIF-1降解反应的限速酶，脯氨酸羟化酶抑制剂可稳定HIF-1的表达。本项目主要研究：（1）脯氨酸羟化酶抑制剂DMOG对MSCs的动员作用与动员来源MSCs的生物学特性；（2）HIF-1 及下游信号通路在介导DMOG动员MSCs中的作用机理。研究结果显示：（1）DMOG对MSCs具有动员作用，能增加外周血及骨髓中MSCs的数量，提高外周血及骨髓中CD45－CD90＋细胞群的比例；（2）DMOG动员外周血来源MSCs阴性表达造血系标记CD34、CD45，阳性表达CD90，具有成神经、成软骨、成骨、成脂分化的潜能，且MSCs经DMOG体内动员或体外处理后仍保留了原有的生物学特性；（3）DMOG在体外对MSCs中HIF-1信号通路具有调控作用，可上调细胞中HIF-1α及其下游SDF-1α、VEGF 蛋白及mRNA的表达水平；（4）DMOG在体内动员MSCs中可上调HIF-1α的表达，且YC-1抑制HIF-1α表达后，外周血与骨髓中MSCs的数量及CD45－CD90＋细胞群比例均减少，动员效率降低，提示HIF-1α在DMOG诱导MSCs动员中起关键作用；（5）DMOG在体内动员MSCs中可上调SDF-1α、VEGF的表达，且SDF-1/CXCR4信号通路经AMD3100阻断，VEGF/VEGFR信号通路经SU5416阻断后，外周血与骨髓中MSCs的数量及CD45－CD90＋细胞群比例均减少，动员效率均降低，提示HIF-1直接调控的下游SDF-1/CXCR4及VEGF/VEGFR信号通路在DMOG诱导MSCs动员中均具有重要作用。本研究结果为寻找MSCs 动员有效途径、建立临床可行的MSCs 动员方法提供了理论与实验依据。