iPSC-MPCs源的外泌体miRNAs调控Smad7/TGFβ通路促受损尿道括约肌细胞外基质重建的作用研究

SUI is a very common disease, especially in women. Our previous studies showed that muscle progenitor cells (MPCs) derived from induced pluripotent stem cells hold great promise for the treatment of stress urinary incontinence (SUI). The therapeutical effects of iPSC-MPCs in SUI are achived by promoting ECM remodeling and improving the reconstruction of urethral sphincter mainly through the paracrine pathway. However, its detailed molecular mechanism is still unclear. Exosomal miRNAs secreted by stem cells is a new and important pathway for intercellular communication. Several studies have showed that exosomal miRNAs play an important role in the stem cell therapy. In our pre-experiment, we have also found that the iPSC-MPCs derived exosome can promote the synthesis of ECM protein in SUI cells, and it contains miRNAs which can regulate the Smad7/TGFβ signaling that is abnormally activated in SUI local microenvironment. Therefore, we propose that iPSC-MPC-derived exosomal miRNAs can regulate the Smad7/TGFβ signaling pathway to promote ECM reconstruction in SUI, and then improve the function of urinary sphincter. In our present project, using SUI animal models and molecular biological techniques, we will further elucidate the role of iPSC-MPC-derived exosomal miRNAs in the treatment of SUI and explore its detailed molecular mechanisms. The completion of this project will promote the clinical translation of our iPSC-MPCs therapy for SUI, and also provide new strategies and targets for the treatment of SUI.

我们前期研究证实iPSC源性肌前体细胞(MPCs)在治疗SUI上具有极大的应用前景，其主要经旁分泌途径促细胞外基质(ECM)重建而改善尿道括约肌控尿功能，但具体分子机制未知。新近研究证实干细胞源外泌体传递miRNA是一种重要旁分泌及再生治疗机制。我们前期预实验亦发现iPSC-MPCs源外泌体可上调SUI细胞ECM蛋白的合成，且其内含有可调控SUI局部组织异常激活的Smad7/TGFβ通路的miRNAs。因此我们提出“iPSC-MPCs源的外泌体miRNAs靶向调控SUI细胞内Smad7/TGFβ通路促进尿道括约肌ECM重建”的假说。本课题中我们将采用整体动物模型和分子生物学实验相结合的方案，深入阐明在iPSC-MPCs促进SUI组织ECM重建中外泌体miRNAs所起的作用及具体分子机制。本项目的完成将促进iPSC-MPCs治疗SUI的临床转化，并为SUI再生医学治疗提供新的策略和干预靶点。

我们前期的研究证实干细胞极其衍生的前体细胞在压力性尿失禁（SUI）上具有极大的应用前景，且其主要通过旁分泌途径促进受损的细胞外基质（ECM）重建而改善尿道括约肌控尿功能，但其中具体分子作用机制未明。为阐明干细胞移植改善尿道括约肌功能具体作用途径，促进干细胞治疗SUI的临床转化，我们开展了本研究。在本项目中我们通过（1）探索骨髓间充质干细胞源性外泌体（BMSC-Exo）体外促人尿道周围原代成纤维细胞ECM代谢的影响，及SUI模型大鼠控尿功能的修护作用；（2）检测MSC-Exo干预后对调控ECM代谢关键性信号通路TGF-β1/Smad关键性分子表达水平的影响；（3）深度miRNA测序、筛选、验证差异表达miRNA，生信预测可能参与TGF-β1/Smad信号通路调控的miRNA，并采用双荧光素酶报告基因系统验证。研究结果揭示：BMSC-Exo不仅能显著改善SUI大鼠的控尿功能，而且促进了受损尿道ECM沉积、平滑肌组织形态学的恢复；分子机制研究上，我们发现BMSC-Exo体内外均能促进组织/细胞内TGF-β1表达水平及Smad2/3的磷酸化水平，但下调了TGF-β1信号通路上游抑制分子Sirt7的表达。miRNA测序从BMSC-Exo中筛选出18个差异表达miRNAs，经生信分析及双荧光素酶报告基因确定外泌体可能通过递送miR-328a-3p负向调控了Sirt7的表达。总之，通过本项目的开展，我们证实BMSC源外泌体通过递送miR-328a-3p抑制Sirt7基因的表达，进而激活 TGF-β1信号通路，调节受损尿道括约肌细胞外机制代谢，最终促进了SUI控尿功能的恢复。本项目中我们通过体内外实验，从组织、细胞和动物模型水平揭示了BMSC源外泌体在SUI上的治疗作用及具体分子机制。我们相信本项目的完成有利于促进干细胞极其相关制剂用于SUI治疗的临床转化，并有可能为SUI的再生治疗提供新的策略和干预靶点。