输注间充质干细胞促进造血重建：TLR2/TLR4及相关miRNA对其疗效的调控研究

The therapeutic benefits of mesenchymal stromal cells (MSCs) for engraftment of hematopoietic stem cells have attracted much attention in recent years. However, the mechanisms that mediate and/or modulate the therapeutic potency of MSCs remain incompletely understood. Our data and recent studies demonstrate that Toll-like receptor 2 （TLR2） and/or TLR4 ligands can activate MSCs, thus modulating their hematopoiesis-supporting role in vitro, differentiation, migration, and immunosuppression capacities. Many factors can influence TLR2/4 ligands released in recipients of hematopoietic stem-cell transplantation (HSCT), indicating MSCs employed in therapy can be potentially exposed to TLR2/4 ligands stimulation. We are interested whether TLR2/4 ligands exposure may modulate MSC therapeutic potential in vivo and what's the underlying mechanism. In this study, we will examine the effects of TLR2/4 activation or inhibition in MSCs on human MSC-mediated hematopoiesis relative role in vitro and human umbilical cord blood transplantation in a NOD/SCID mouse model. Secondly, we will analyze and confirm the expression profile of microRNAs (miRNAs) , a class of novel regulatory RNAs that play an important role in regulating gene expression , in TLR2/4 ligands-primed human MSCs by microarray and real time RT-PCR. Over-expression and knock-down approaches will be used to determine the effects of selected miRNAs on MSC-mediated hematopoiesis relative function after TLR2/4 exposure. The potential target genes will be selected by bioinformatic prediction and experimentally validated. Exploring the genes and pathways regulated by these miRNAs will help us to understand the detailed molecular mechanisms and signaling pathways of TLR2/4 on modulation of MSC-mediated hematopoiesis relative role. Based on our study, we hope to find some potential targets for enhancing therapeutic potency of MSCs in HSCT.

输注间充质干细胞（MSC）促进造血重建是造血干细胞移植（HSCT）的研究热点之一，但调控其疗效的机制未明。我们的研究和已有的报道显示：Toll样受体2（TLR2）和/或TLR4能调节MSC的体外造血支持功能、迁移、分化及免疫抑制能力等。HSCT时，多种因素均可影响受者体内TLR2/4配体的释放。对此我们的问题是：MSC是否因暴露于TLR2/4配体而影响疗效？它们的分子机制如何？为此我们拟利用NOD/SCID小鼠人造血干细胞移植模型和体外实验，研究活化和阻断MSC上TLR2/4对造血重建的影响及其机制；并借助miRNA芯片和PCR技术寻找TLR2/4活化相关的miRNA，通过抑制或过表达相关miRNA，研究它们对MSC介导的造血相关功能的调控，筛选验证其调控的靶基因和靶蛋白，进而分析TLR2/4调控MSC相关功能所涉及的分子信号通路。项目的实施可能为提高MSC的疗效寻找到新的靶点。

输注间充质干细胞（MSC）促进造血重建是造血干细胞移植（HSCT）的研究热点之一，但调控其疗效的机制未明。HSCT时，多种因素均可影响受者体内TLR2/4配体的释放。本课题中我们利用体外实验和NOD/SCID小鼠人造血干细胞移植模型，研究活化和阻断MSC上TLR2/4对造血相关功能的影响及其机制；并借助miRNA芯片和PCR技术寻找TLR2/4活化相关的miRNA，通过抑制或过表达相关miRNA，研究它们对MSC介导的造血相关功能的调控，筛选验证其调控的靶基因和靶蛋白，进而分析TLR2/4调控MSC相关功能所涉及的分子信号通路。我们的研究结果显示：Toll样受体2（TLR2）和/或TLR4能调节MSC的迁移、黏附、分化及体外造血支持功能等；并利用NOD/SCID小鼠人脐血CD34+ 造血干细胞和人骨髓MSC共移植模型，证实TLR2和TLR4活化的MSC能增强CD34+细胞体内归巢和加速重建造血。进一步研究显示;TLR2和TLR4诱导的骨髓间充质干细胞中的miRNAs表达存在差异,且多个miRNAs可调控MSC的多种生物学功能；例如：miR-146a对BM-MSCs成脂分化有促进作用并可调控MSC的造血支持功能，miRNA-99a-5p对BM-MSCs的成骨分化有促进作用，miR-214对MSC诱导CD34+细胞迁移起到促进作用，而miR-155通过靶向MYLK抑制BM-MSCs的迁移。项目的实施为提高MSC的疗效寻找到可能的新靶点。