基于Bach1调控血管新生分子机制的梗死心肌组织血管化治疗与实验研究

Cell transplantation provides a new method and idea for treating the myocardial infarction. However, therapeutic effect of the transplantation is limited. The key problem is the lack of blood vessels in the ischemic myocardial tissue limits the level of transplanted cell survival and physiological functioning, which should be solved immediately. The micro-vascularize strategy composite endothelial cells can be more effective in improving the ischemic microenvironment, but the lack of the sources of endothelial cells and a low efficiency of the vascularization seriously reduce the effect of micro-vascularization. Our preliminary research is the first study to show Bach1 is the negative regulator factor of angiogenesis, the angiogenesis efficiency of endothelial cells can be significantly improved when the Bach1 expression is down-regulated. Therefore, this project through different trans-differentiation programs seeks to resolve the problem of endothelial cell-derived, Bach1 in endothelial cells will be silenced to improve the ability of vascularization and the efficiency of angiogenesis. Three-dimensional cell culture in vitro to observate the angiogenesis potential when Bach1 down-regulated in endothelial cells. Then mix the endothelial cells whose Bach1 is down-regulated with hydrogel to treat the tissue for micro-vascularization after myocardial infarction. Meanwhile, establishment the Bach1 complete knockout mice and the myocardial tissue specificity Bach1 conditional knockout mice, using their AMI model to evaluation the role of Bach1 with impacting the micro-vascularization of infarcted myocardial tissue and cardiac function in vivo. This project has important scientific significance and clinical application for the treatment of post-infarction and myocardial micro-vascularization of tissue regeneration.

细胞移植为心肌梗死治疗提供了新思路，但疗效有限。归因于贫乏的血管化水平限制着移植细胞的存活与生理功能发挥。复合内皮细胞的微血管化策略可有效地改善缺血微环境，但内皮细胞来源不足及成血管效率低又严重降低组织的微血管化效果。我们前期研究首次发现Bach1负性调控血管新生，下调Bach1表达可显著提高内皮细胞的血管新生效率。本项目试图通过成纤维细胞转分化方案解决内皮细胞来源问题；对内皮细胞进行Bach1基因沉默，提高其成血管能力及血管新生效率；体外三维细胞培养观察Bach1表达下调的内皮细胞在血管新生方面的潜力；合并水凝胶载体进行梗死后心肌组织的微血管化治疗。建立Bach1完全性基因敲除鼠以及心肌组织特异性Bach1条件性基因敲除鼠，利用其AMI模型在体评价Bach1下调对梗死心肌血管化的作用和其对心功能的改善效果。本项研究将对梗死后心肌的血管化及组织再生治疗具有重要的科学意义和临床应用价值。

Bach1会阻碍Wnt/β连环蛋白信号通路，抑制内皮细胞沿管腔的增生、迁移，并抑制手术诱导的后肢缺血小鼠模型（HLI）的血管再生。但是Bach1在斑马鱼血管发生发展过程中的作用尚不清楚。本实验通过在体观察外源导入Bach1 mRNA，观察过表达的Bach1b对斑马鱼Wnt/β连环蛋白信号通路和血管发生过程的影响。我们发现Bach1活跃表达在斑马鱼早期胚胎发育的全过程，过表达的mRNA导致斑马鱼胚胎发育过程中体节间血管ISV和背长轴血管DLAV都没有很好地形成，并且抑制了斑马鱼胚胎阶段由Wnt/β连环素信号与Wnt8a协同刺激的VEGF和IL-8的基因表达。同时，我们取人脐静脉内皮细胞HUVECs试图通过离体实验探究Bach1调控Wnt信号靶基因VEGF表达的机制。染色质免疫沉淀ChIP 的结果提示Bach1占据了HUVECs 的VEGF启动子上TCF/LEF的结合位点。Bach1通过把HDAC1招募到VEGF启动子来抑制HUVECs的VEGF转录。同时外源性注射VEGF或IL-8可以一定程度上抵抗Bach-1驱使的抗血管生成作用。综上所述，Bach1阻碍了斑马鱼胚胎血管形成发育的过程，并且这种功能也与Wnt/β连环蛋白信号途径和VEGF、IL-8的表达衰减有关。