靶向TGF-β1/Smads通路实现梗死心肌“适宜”硬度的时域延展实验研究

Myocardial fibrosis is the pathological basis of myocardial stiffness after infarction. TGF-β1/Smads signaling is one of the most important molecular pathway modulating myocardial fibrosis process. Our previous research indicated that the “suitable” stiffness of infarcted myocardium (42kPa, 1 to 2 weeks post myocardial infarction [MI]) provided a optimized physical microenvironment for the survival and specification of transferred cells. However, the narrow time domain of “suitable” stiffness significantly restricted the efficacy of cell-based cardiac repair. We presumed that optimal time frame of “suitable” stiffness of infarcted myocardium might be expanded by targeting TGF-β1/Smads signaling pathway. The study would investigate the change in time domain of “suitable” stiffness of infarcted myocardium following targeting TGF-β1/Smads pathway in Balb/c mouse MI models. Moreover, myocardial extracellular matrix (ECM) was extracted by the method of decellularization of infarcted myocardium and the impact of ECM composition on infarcted myocardial stiffness was estimated. Polyacrylamide gel substrates with varied stiffness were used to mechanically mimic the stiffness of infarcted myocardium in vitro. Bone marrow-derived CD34+ cells were plated and cultured on the flexible culture substrates and on fibroblast-derived ECM after targeting TGF-β1/Smads. the relevant factors influencing “suitable” stiffness of infarcted myocardium and their pro-differentiation effect on CD34+ cells along endothelial cell lineage would be elucidated. The proposal will contribute to a better understanding of key cellular and molecular mechanisms underlying the “suitable” physical microenvironment within infarcted myocardium, and subsequently provide effective interventional targets and therapeutic strategy for widening “time window” of effective cell treatment post MI.

心肌梗死（MI）后心肌纤维化是决定梗死心肌硬度的病理基础，TGF-β1/Smads是调控心肌纤维化的重要通路。前期研究证实，梗死心肌“适宜”硬度（约42kPa，MI后1-2周）为骨髓源细胞的生存和分化提供最佳物理微环境。然而，较窄的“适宜”硬度时间窗，严重制约细胞移植疗效。本项目提出“靶向TGF-β1/Smads通路实现梗死心肌‘适宜’硬度时域延展”的科学设想。拟借助MI小鼠TGF-β1/Smads通路的靶向阻断，采用原子力显微镜及心脏去细胞技术，阐明梗死心肌“适宜”硬度的时域变化及细胞外基质（ECM）的组分贡献；借助聚丙烯酰胺凝胶实现心肌硬度的体外模拟，结合靶向干预后心脏成纤维细胞源ECM沉积，开展骨髓CD34+细胞培养，探讨“适宜”硬度的决定因素及核心环节对CD34+细胞分化的诱导作用。本项目将揭示MI后心肌“适宜”硬度相关信号分子的调控机制，为拓宽细胞治疗“时间窗”提供有效干预靶点。

纤维化是决定组织硬度的病理基础，TGF-β1/Smads是调控纤维化的重要通路。“适宜”的基质硬度为移植细胞的生存和分化提供最佳物理微环境。本项目研究发现1、在基质定植和向内皮分化方面，CD34+内皮祖细胞优于CD34-内皮祖细胞，42KPa是CD34+内皮祖细胞定植和向内皮细胞分化的最“适宜”基质硬度。2、通过靶向抑制TGF-β1/smad通路改变细胞外基质组分，可增加间充质干细胞在细胞外基质上的定植，减少间充质干细胞在氧化应激条件下的凋亡。3、白介素11是TGF-β1促进成纤维细胞表型转化及细胞外基质产生的主要效应分子，白介素11可通过其受体及其下游STAT3信号通路促进间充质干细胞生长、增殖、迁移和减少间充质干细胞在氧化应激条件下的凋亡，其次，白介素11可提高间充质干细胞在缺血组织的移植疗效，促进缺血下肢的血流恢复。本项目研究结果表明，通过干预细胞外基质组分及其下游分子，影响基质硬度，有利于改善间充质干细胞治疗疗效，为细胞治疗提供最佳移植物理微环境提供了重要的干预策略。