通心络联合Angiopoietin-1基因修饰改善MSCs移植微环境及心肌梗死后心肌重构的作用和机制

Stem cells transplanting and therapeutic angiogenesis have been the hot research areas of myocardial revascularization after myocardial infarction. But it is harmful for stem cells to homing, differentiation and multiplicationin the ischemic microenvironment. The low survival rate of stem cells in the ischemic myocardium microenvironment is the bottle neck in treating ischemic diseases with stem cell transplantation .So the microenvironment remolding becomes the key to determine the therapeutic effect of stem cell transplantation after myocardial infarction. Angiopoietin-1 can not only promote angiogenesis and blood vessel maturity, but also improve the structure reconstruction of blood circulation after myocardium infarct. However expression of angiopoietin-1 was down regulated in the ischemic myocardium. Meanwhile, Tongluo prescription - tongxinluo capsule can dredge the blood vessel and improve microcirculation to promote the functional recovery of of blood circulation. Both of them can improve the transplanting microenvironment and increase the chances of survival of the transplanting stem cells survive and differentiation. The study is to use adenovirus carrier of coding angiopoietin-1 gene to transfect autologous mesenchymal stem cell for transplantation into the infarcted myocardium in miniature pig via coronary artery. Expression of angiopoietin-1 and the treatment of tongxinluo capsule will promote the structure reconstruction and functional recovery of blood circulation in infarcted myocardium, and improve the transplanting microenvironment and motivate homing, differentiation and proliferation of the transplanting stem cells, and enhance the structure reconstruction and function recovery of myocardium after myocardial infarction. This Lab will observe the effect of angiopoietin-1 and tongxinluo capsule on the migration and differentiation of MSCs and the expression of SDF-1, CXCR-4, VCAM-1, SOD et al, which can probe into its mechanism of improving the transplanting microenvironment. The study will provide the basis for stem cells transplantation and remolding of transplanting microenvironment after myocardium infarct on the basis of integrated traditional and western medicine,as a whole and local treatment.

干细胞移植和治疗性血管生成已成为心肌梗死后心肌重建的研究热点，但梗死后缺血微环境却不利于移植干细胞的归巢、分化和增殖，微环境重建成为决定干细胞心脏移植疗效的关键。Angiopoietin-1可促使血管生成和成熟，促使梗死心肌血运结构重建，缺血时却明显下调，通络方剂通心络可疏通血络，促使血运功能重建，两者均可改善移植微环境，并促使移植干细胞存活和分化。课题拟以编码Ang-1基因腺病毒载体转染自体MSCs经冠脉移植小型猪心肌梗死区域，通过表达Ang-1并联合通心络治疗，"里应外合"以促进梗死心肌血运结构和功能重建，改善移植微环境并促使移植干细胞归巢、分化和增殖，促进心肌梗死后心肌重建和功能恢复，并观察Ang-1和通心络对MSCs迁移，分化以及SDF-1、CXCR-4、VCAM-1、SOD等表达的影响以探讨其改善移植微环境的机制。课题将为中西医结合基础上的干细胞心脏移植以及移植微环境改造提供依据

心力衰竭发生机制的关键是心肌重构，改善心肌重构是心血管临床和基础的重大挑战。本研究目的在于探讨MSCs联合通心络改善梗死心肌重构的作用和机制。.已发现：.1.成功制备的编码Angiopoietin-1和VEGF165基因的质粒，以Angiopoietin-1重组质粒转化CHO细胞株后行DNA测序和鉴定。.2.成功培养MSCs及诱导向心肌细胞转化。移植到大鼠梗死心肌的MSCs能顺利存活，但转染pSecTag-Ang1后的MSCs移植到心肌组织中存活率很低。同时发现缺氧干预的心肌细胞培养液可诱导MSCs向心肌细胞转化。.3.缺氧环境下心肌细胞发生缝隙连接重构和Cx43下调，缺氧导致心肌细胞凋亡和Cx43下调与AT1受体信号通路有关。压力超负荷大鼠心肌Cx43表达下调，并可导致心肌电生理重构。.4.通心络可以抑制心肌细胞的肥大并改善缝隙连接重构和Cx43下调。.5.缺氧环境下MSCs和心肌细胞的硫化氢生成下调。通过建立急性心肌梗死大鼠模型，我们证实心肌梗死大鼠心肌组织中的硫化氢代谢酶3-MST和CSE均出现明显下调。同时临床观察中发现冠心病患者硫化氢生成下调和同型半胱氨酸水平上调。.6.Ang-1质粒联合MSCs不仅可改善心肌梗死大鼠心肌重构和心功能，改善梗死心肌电生理重构和心肌纤维化，而且可以改善梗死心肌的血管新生。.7.通心络可以改善心肌梗死后心肌重构和血管新生，其机制可能与促进H2S生成有关。实验还发现通心络可明显上调梗死心肌的硫化氢合成酶蛋白的表达。.8.通心络和硫化氢可通过上调血管生成相关因子促进血管新生。研究发现通心络可以上调VEGFR2和EPHA2的表达，并与硫化氢调控机制有关。实验还发现硫化氢可以上调糖尿病大鼠心肌Ang1等血管生成因子的表达，促进血管新生。 .9.鉴于发现硫化氢信号通路参与通心络改善移植微环境和干细胞归巢分化的机制，课题组在基金的支持下开展H2S改善心肌重构及信号调控机制的相关研究，目前发现硫化氢可通过下调PPAR_γ、NF-KB、TGF-β、PKC等信号通路减少氧化应激和细胞自噬而改善MMPs/TIMPs失调和心肌纤维化，同时硫化氢还可下调PI3K/AKT1并抑制内质网应激，还可改善缝隙连接重构和心肌电生理重构。. 以上有望为心肌重构的临床防治以及机制探讨提供进一步依据。