Tβ4稳定HIF-1α功能的信号通路及其促进大鼠心梗后血管新生的作用机制研究

Coronary heart disease (CHD) has become one of the most serious diseases to jeopardize human health in recent years in our country. But present traditional treatments for CHD such as medicine, interventional therapy or surgery have some disadvantages. Our previous experiment has confirmed that transplantation of bone marrow stromal cells (MSCs) transferred with vascular endothelial growth factor (VEGF) into myocardial infarction area can promote angiogenesis and improve cardiac function. Because the new blood vessels induced by a single growth factor have the problem of instability and leakage, we then tried to transferred Hypoxia-inducible factor (HIF) gene into MSCs to adjust its downstream growth factors acting together to generate mature and stable neovascularization by promoting the expression of HIF. However, we also found the expression of HIF was time limited. Thymosin β4 (Tβ4) is a powerful stimulating factor for coronary vessel generation and stability. It was showed that Tβ4 can improve the expression of VEGF and promote angiogenesis by stabilizing HIF-1α, but its exact mechanism is unclear. So here we plan to use exogenous Tβ4 intervening MSCs to observe its effect to HIF-1α and explore its possible mechanism. Simultaneously, we transplant MSCs transferred with Tβ4 gene into rat myocardial infarction area to observe the relation between Tβ4, HIF-1α and VEGF, and examine its effect to angiogenesis in infarcted area and cardiac function after myocardial infarction. This may be helpful for Tβ4 treatment of coronary heart disease by providing a theory basis and experimental foundation.

目前冠心病的各种治疗方法均不十分理想。我们前期研究证实血管内皮生长因子（VEGF）转染骨髓间充质干细胞（MSCs）移植入心梗区可促进血管新生和改善心功能，为避免单一生长因子作用生成的血管存在不稳定和易渗漏问题，进一步将缺氧诱导因子（HIF）转染MSCs，通过促进HIF表达来调节下游多种生长因子共同作用以生成成熟稳定的新生血管，然而我们同时发现HIF表达存在时效较短的问题。胸腺素β4（Tβ4）是强有力的促进冠脉生成和稳定的刺激因子，有研究表明Tβ4可通过稳定HIF-1α来影响VEGF的表达而促进血管新生，然而其具体机制不明。本课题拟通过外源性Tβ4干预MSCs，观察其对HIF-1α表达的调控作用并探讨可能的机制，同时将转染Tβ4的MSCs移植入大鼠心梗区，观察体内其与HIF-1α和VEGF之间的关系，以及对心梗区血管新生的作用和心梗后心功能的影响。为Tβ4治疗冠心病提供理论依据和实验基础。

目的：已知肌动蛋白螯合蛋白胸腺素β-4（Tβ4）和缺氧诱导因子（HIF）-1α参与心肌梗死后的血管生成。本文旨在探讨Tβ4诱导HIF-1α的机制，探讨转染Tβ4基因（TMSB4）的骨髓间充质干细胞（BMMSCs）对大鼠心肌梗死模型的影响。.方法：分离培养大鼠骨髓间充质干细胞,，将TMSB4基因采用慢病毒介导法一起转染间充质干细胞。通过结扎前降支，完成心肌梗死模型。将心肌梗死大鼠随机分为3组（每组12只），1周后，在心脏梗死边界区注射TMSB4过表达的BMMSCs（BMMSC-TMSB4OE），TMSB4水平正常（BMMSC- TMSB4WT）或细胞培养介质（MI），第四组动物（n = 12）接受建立心肌梗死所需的所有外科手术除了结扎前降支。注射后4周，通过超声心动图测量心脏功能。通过TTC染色计算梗塞面积。通过CD31免疫荧光组织化学评估受损心脏区域中的血管生成。进行体外实验，观察外源性Tβ4对HIF-1α的影响，探讨其可能的作用机制。.结果：体内实验显示，处理后第4周的血管密度在BMMSC-TMSB4OE处理中比在BMMSC-TMSB4WT处理的动物中高约2倍（p <0.05）。相较于对照组，两个细胞治疗组心脏的心功能和梗塞面积均显着改善，其中BMMSC-TMSB4OE治疗组的动物改善更加明显（各自p <0.05）。在体外，HIF-1α和磷酸化的HIF-1α被外源性Tβ4大大增强，并被HIF抑制因子（FIH)YC-1阻断。.结论：我们的数据表明，TMSB4转染的BMMSCs可显着改善心肌缺血的恢复，通过ERK通路促进HIF-1α磷酸化，同时通过FIH 通路抑制磷酸化HIF-1α的降解。