间充质干细胞外泌体miRNA调控放射性肠损伤致病细胞γδT17的发育、功能及表型重塑的作用机制

Mesenchymal stem cell (MSC)-derived exosomes contain various miRNAs, which are capable of regulating the development of T cells and their function. Herein, MSC is capable of inhibiting the differentiation of CD4+ Naive T cells into functional Th17 cells. Besides, MSC can up-regulate the expressions of Foxp3 and IL10 genes by functional Th17 cells. There are similarities between γδT17 and Th17 cells in their development and function. To date, it is still not found any report associated with the regulation of γδT17 development, function or phenotype remodeling by the MSC. In preliminary study, we found that the pathogenic cells of radiation-induced intestinal injury are γδT17 cells, which derive from CD4 and CD8 double-positive (DP) thymocytes, and that irradiated MSC distinguishes its miRNA-expressing profile from the normal MSC. Basing on these results, we intend to investigate the specific roles of MSC-derived exosomes in regulating the biological processes associated with γδT17 development, function and phenotype remodeling. Moreover, we plan to explore the mechanisms by which some key exosomal miRNAs, determined by the theoretical model of Waddington landscape, regulate the γδT17 development, function and phenotype remodeling. Thereafter, we will explore the mechanism of the differential expression of miRNAs by the irradiated MSC, which is somewhat regulated by ATM-KSRP-mediated miRNA splicing. We aim at demonstrating that normal MSC can inhibit the development of γδT17 cells from their progenitors, and can confer the anti-inflammatory phenotypes of Foxp3 and IL10 to γδT17 cells. Moreover, the mechanisms of some key exosomal miRNAs in regulating γδT17 development, function and phenotype remodeling should be determined. The prospective results of this study will provide valid evidences for management of radiation-induced intestinal injury by using MSC-therapy.

间充质干细胞（MSC）的外泌体miRNA能调控T细胞的发育及功能。其中，MSC能抑制CD4+新生T细胞向Th17发育，还能上调Th17表达Foxp3和IL10基因。γδT17与Th17的发育及功能相似。目前，MSC对γδT17发育、功能及表型重塑的调控作用未被报道。我们发现：放射性肠损伤致病细胞γδT17来源于DP期胸腺细胞，并且受照的MSC差异表达miRNA。我们据此研究：1）MSC外泌体对γδT17发育、功能及表型重塑的调控作用；2）经能量地貌理论模型确定的高权重miRNA对调控γδT17发育、功能及表型重塑的机制；3）ATM-KSRP介导的miRNA剪切对调控受照MSC差异表达miRNA的机制。目标：①明确未受照的MSC能抑制γδT17发育，还能促进γδT17获得Foxp3+/IL10+抗炎表型；②掌握高权重外泌体miRNA在目标①中的作用机制。预期为MSC治疗放射性肠损伤提供依据。

本项目在前期研究基础上发现γδT17细胞可能是放射性肠损伤的致病细胞之一。另外，我们前期发表的研究工作也证实间充质干细胞能够修复放射性肠损伤。鉴于间充质干细胞强大的免疫调节能力，本项目中拟研究间充质干细胞在修复放射性肠损伤过程中，对γδT17细胞发育及表型重塑的作用机制。我们发现：间充质干细胞外泌体中存在较多数量的miRNA。其中，我们的研究发现了miRNA1246是间充质干细胞发挥主要效能的微小RNA类型。通过功能学实验我们证实了一定水平的辐射能够上调间充质干细胞外泌体miRNA1246的表达；同时，还能够加速辐射诱导的DNA损伤修复。此外，在“势”与“流”能量地貌模型构建方面，通过随机波动环境中生态进化系统的全局稳定性和动力学性质，将非平衡态“势”与“流”能量地貌理论应用于分析细胞系统模型的全局稳定性和拓扑结构，并得到这个系统的能量地貌和几率流。这一模型的建立为解释细胞分化及发育过程中能量的变化提供物质基础。在放射性肠损伤发病机理方面，我们揭示了不同水平的辐射剂量能够差异地改变小鼠肠道菌群结构，并且在特定菌群结构的影响下，参与构成损伤微环境的免疫学特征性细胞类型势不同的。例如，在亚致死剂量（7.5 Gy）全腹照射下，小鼠小肠派氏斑以滤泡辅助T细胞浸润和B细胞激活为特点；而在致死剂量（15 Gy）全腹照射下，派氏斑中以产IL-17的免疫细胞为主要特征，这表明了不同水平的辐射通过差异地改变肠道菌群结构对特定条件下的免疫微环境的塑造能力具有差异。在此基础上，接受相同水平辐射的直肠癌患者，其肠道菌群结构在被辐射重塑后也是不同的。其中，一部分以高丰度得产丁酸盐细菌为特征；而另一部分以高丰度得致炎细菌为特征。这种差异也一定程度影响了其原发肿瘤对辐射的敏感性。另外，将对辐射抵抗的直肠癌患者的粪菌移植入小鼠模型中能够诱导Th17细胞在结肠中浸润。总的来说，我们的研究从放射性肠损伤的损伤的发病机制及损伤修复两大角度分别提供了新的数据，为相关疾病得防治提供新思路。