RhoE表达失调对心肌梗死后移植c-Kit+/Nkx2.5+骨髓间充质干细胞生物学功能的影响及机制

The adaptive response of transplanted stem cells to local hypoxia is crucial for its biological function after myocardial infarction (MI). To date, it is still not clear about the mechanisms regarding endogenous molecule-regulated hypoxia-indictable factor 1 α (HIF-1α) expression and activity, which is critically involved in the pathophysiology of MI. Interestingly, we have previously found that c-Kit+/Nkx2.5+ bone marrow mesenchymal stem cells (BMSC), a special subsets of stem cells, were involved in the recovery of cardiac function via HIF-1α-mediated activation; besides, HIF-1α can directly regulate the expression of its potential downstream gene RhoE, and the binding of RhoE protein and HIF-1 protein thereafter inhibits the degradation of HIF-1α via UPS system. In this proposal, we hypothesize that there is a HIF-1α/RhoE feedback regulation system after MI, which is critically involved in the activation of stem cells. The dysfunction in the HIF-1α/RhoE system would lead to deterioration in the biological function of transplanted stem cells post MI. In this project, we plan to isolate RhoE wild type c-Kit+/Nkx2.5+BMSC (BMSC-WT). At the same time, the RhoE knockout c-Kit+/Nkx2.5+ BMSC (BMSC-KO) and RhoE-overexpressing c-Kit+/Nkx2.5+ BMSC (BMSC-OV) are established through the CRISPR/Cas9 method. Using these three BMSC cells, the establishment, function and mechanism about HIF-1α/RhoE signal feedback system post hypoxic stimuli are analyzed in vitro, and the effect of targeting HIF-1α/RhoE signal on the biological function of transplanted BMSC after MI was investigated in vivo. The implementation of the project would provide a novel mechanism about HIF-1α-activated stem cells post MI, and promote the progression of translational medicine regarding treatment of MI based on stem cell transplantation.

干细胞对心肌梗死（MI）后低氧反应的适应对其生物学功能至关重要但调节机制不明。我们前期发现MI后c-Kit+/Nkx2.5+BMSC在HIF-1α的激活下促进心功能恢复；MI后HIF-1α潜在靶基因RhoE表达下调，而RhoE蛋白与HIF-1α蛋白结合可抑制HIF-1α的降解。本项目假设MI后存在HIF-1α/RhoE反馈调节系统，该系统的异常影响了MI后移植干细胞的活化。项目拟分离RhoE野生型c-Kit+/Nkx2.5+BMSC，同时基于CRISPR/Cas9系统分别构建RhoE敲除和过表达c-Kit+/Nkx2.5+BMSC，以此三个BMSC为模型，体外实验分析低氧后HIF-1α/RhoE信号反馈系统的建立、作用及工作原理，体内实验探讨靶向HIF-1α/RhoE信号对MI后移植BMSC生物学功能的影响。项目的实施将提出HIF-1α活化干细胞的新机制，促进干细胞治疗MI转化医学的进展。

低氧刺激对移植干细胞治疗缺血性心脏病有益但获益机制仍不明确，本项目假设心肌缺血缺氧条件下通过局部的HIF-1α/RhoE反馈调节系统来活化移植的干细胞。项目研究了⑴低氧刺激对干细胞分化的影响与机制；⑵基于CRISPR/Cas9技术编辑H9C2中RhoE基因，基因芯片筛查差异表达基因并进行相关的生信分析；⑶基于CRISPR/Cas9技术体外编辑大鼠c-Kit+ BMSC中RhoE基因；⑷基于CRIPSR/Cas9技术构建RhoE基因敲除的SD大鼠；⑸RhoE基因表达缺失对c-Kit+ BMSCs一般生物学功能的影响与机制分析；(6)RhoE对巨噬细胞极性化和在心肌梗死修复中的作用。研究发现，⑴低氧刺激可通过HIF-α/Erk/Jagged1/Notch1信号促进干细胞的心肌谱系分化，也可诱导SRF-N表达而抑制心肌细胞谱系分化；⑵基于CRIPSR/Cas9技术成功实现了对心肌细胞H9C2中RhoE基因的编辑，筛选了RhoE表达改变后的差异基因，富集了相应的生物学功能和信号途径；⑶体外基于CRISPR/Cas9技术编辑BMSC中RhoE基因有难度，细胞得率低；⑷首次基于CRISPR/Cas9技术获得了RhoE基因敲除的SD大鼠，成功分离到相应的c-Kit+ BMSC；⑸无论是过表达HIF-1α还是物理性低氧（2% O2）刺激均抑制了c-Kit+ BMSC中RhoE的表达，继而增加了ROCK1活性；BMSC中HIF-1α与RhoE并不存在反馈调节网络；⑹RhoE表达缺失通过ROCK1信号活化而促进细胞的增殖，但抑制了其迁移、旁分泌促血管生成因子和心肌谱系分化；⑺RhoE表达缺失促进巨噬细胞的增殖，有利于M1型极化而发挥促炎作用。研究结果证实，局部低氧微环境对干细胞的心肌谱系分化发挥促进和抑制的双向调节作用；RhoE基因与心血管疾病关系密切；局部低氧条件下RhoE基因表达减少后对干细胞的一般生物学特性产生了不利的影响，并产生促炎反应；RhoE不足与HIF-1α无直接调控关系，提示存在其他的导致RhoE表达下调的机制。本项目的研究结果丰富了干细胞治疗缺血性心肌病的理论体系。