我们前期系列研究证实心肌梗死（MI）后低氧诱导因子1α（HIF-1α）在活化心脏干细胞（CSC）参与心功能恢复过程中起关键作用，但HIF-1α介导CSC活化的机制不明。RhoE为ROCK1信号内源性抑制子。我们发现HIF-1α可直接调控RhoE的表达，且RhoE蛋白与HIF-1α蛋白物理性结合后抑制了HIF-1α的泛素化降解。本项目假设MI后可能存在HIF-1α/RhoE反馈调节系统，该系统的激活参与了MI后CSC的活化而介导了心功能的恢复。本项目拟分离RhoE野生型CSC，同时基于CRISPR/Cas9系统构建RhoE敲除CSC和RhoE过表达CSC，以此三个CSC细胞系为模型，体外分析低氧后HIF-1α/RhoE信号反馈系统的建立及工作原理，体内实验探讨HIF-1α/RhoE信号对MI后CSC活化的作用。项目的实施将提出HIF-1α活化CSC的新机制，促进CSC治疗MI转化医学的进展。

Our serial previous investigations have proved that myocardial infarction (MI)- activated hypoxia-inducible factor 1 (HIF-1α) may play a critical role in the driving the cardiac stem cells (CSCs) to participate in the cardiac function recovery post MI. However, the mechanisms about HIF-1α-mediated activation of CSCs need to be further clarified. RhoE, also named as Rnd3, is an endogenous inhibitor for ROCK1 signal which hyperactivated post MI. Recently, we have found that HIF-1α could directly mediate RhoE expression, and RhoE protein physically binds with HIF-1α protein hereafter attenuated the degradation of HIF-1α through ubiquitination system (UPS). Therefore, we hypothesize there may be a HIF-1α/RhoE feedback system, which contributes to the recovery of cardiac function post MI through activating the CSCs. In this proposal, we intend to set up the RhoE gene wild type CSCs (CSC-WT), At the same time, the RhoE gene knockout CSCs (CSC-RhoE-/-) and overexpression CSCs (CSC-RhoE-OV) were constructed through transfection the CSC-WT with plasmids based on CRISPR/Cas9 genomic editing system. Using these three CSC cell lines, we seek to investigate the establishment and molecule mechanism of HIF-1α/RhoE signal feedback system in vitro, and the contribution of HIF-1α/RhoE signal in the CSC activation post-MI in vivo. The implementation of the project would provide a novel mechanism about HIF-1α-activated CSC post MI and promote the progression of translational medicine regarding treatment of MI based on CSC transplantation.

心肌梗死（MI）后干细胞移植成功率低是一个亟待解决的难题。本项目假设MI后局部移植干细胞中由于RhoE的丢失从而不能稳定HIF-1α这一重要的保护因子，使得干细胞不能有效活化而影响其生物学功能。本研究从c-Kit+骨髓间充质干细胞（c-Kit+ BMSC）为对象，从以下两个体外研究内容开展工作:(1) RhoE 野生型BMSC（BMSC-WT）和RhoE基因敲除BMSC（BMSC-RhoE-/-）细胞的获取；(2)分析体外低氧刺激对BMSC-WT 和BMSC-RhoE-/-一般生物学特性的影响。结果发现：(1)应用MACS 法成功从成年雄性SD大鼠BMSC中分离c-Kit+亚群，即为c-Kit+ BMSC-WT。(2)采用基于CRISPR/Cas9 技术的基因编辑，成功筛选出针对SD大鼠RhoE基因有效的敲除靶点，包装出滴度为3×10^8 TU/ml的慢病毒颗粒。(3)采用慢病毒感染BMSC-WT，经筛选后成功获得BMSC-RhoE-/-。(4)以常氧为对照，体外低氧（2%O2）培养BMSC-WT可明显刺激其增殖，表现为相应的细胞周期S期增加，G1期减少。(5)ROCK1靶向抑制剂法舒地尔（Fasudil）明显促进低氧和常氧下BMSC-WT的增殖，低氧联合Fasudil较之常氧联合Fasudil更显著促进BMSC-WT增殖，提示ROCK1信号对BMSC增殖起重要作用，也意味着BMSC-RhoE-/-较BMSC-WT增殖能力更强。(6)设计了针对大鼠HIF-1α的过表达质粒，并进行特定氨基酸位点突变以提高其在常氧条件下的稳定性但不改变其活性，成功包装出相应的高滴度慢病毒颗粒。(7)过表达HIF-1α明显抑制BMSC-WT中RohE表达。(8)物理性低氧（2%O2）减低BMSC-WT中RhoE的表达，并下调p-MCL2/MCL2比值进而减低RohE的活性。(9)与常氧对比，低氧（2%O2）促进BMSC-WT的迁移。(10)与常氧对比，低氧（2%O2）促进BMSC-WT分泌VEGF及bFGF因子。我们的结果初步证实:(1)基于CRISPR/Cas9 系统的基因编辑系统可用于大鼠BMSC的基因改造；(2)低氧促进干细胞的增殖、迁移及旁分泌能力；(3)内源性ROCK1信号对干细胞的增殖具有抑制作用。后期将进一步完善对BMSC-RhoE-/-生物学特性的分析。