超声靶向纳泡破坏介导Gal-3shRNA转染抗心肌纤维化和左室重构的研究

Myocardial fibrosis and Left ventricular remodeling (LVR) are the main causes that make heart failure difficult to cure. Studies have shown that galectin-3 (Gal-3) is an important factor leading to myocardial fibrosis and LVR. Inhibition of Gal-3 can prevent myocardial fibrosis and LVR. Introducing exogenous shRNA can inhibit the expression of Gal-3. However, how to efficiently transfer shRNA into cardiomyocytes is the key. Previously, ultrasound targeted microbubble destruction mediated shRNA gene transferring into vascular endothelial cells could promote vascular growth and effectively treat myocardial ischemia; however, microbubbles could not penetrate the vessel wall, so it was difficult to transfer myocardial cells. . Based on the above theories and foundations, this project intends to prepare ultrasound-targeted nanobubbles to mediate Gal-3 shRNA transfection to inhibit myocardial fibrosis and prevent LVR. The main research contents include: (1) preparation of nanobubbles highly encapsulated with Gal-3 shRNA; (2) detection of transfection efficiency and silencing effect of ultrasound-targeted nanobubbles destruction mediated Gal-3 shRNA under different parameters in vitro; (3) preparation of LVR rat model and evaluation of ultrasound-targeted nanobubbles. Vesicle destruction mediates the anti-LVR effect of Gal-3 shRNA transfection into cardiomyocytes, and studies its mechanism. The implementation of this project can provide new ideas for the treatment of heart failure.

心肌纤维化和左室重构（LVR）是心衰难治的主要原因。研究表明，半乳凝素-3（Gal-3）是导致心肌纤维化和LVR的重要因子，抑制Gal-3可阻止心肌纤维化和LVR。导入外源性ShRNA可抑制Gal-3表达。然而如何高效转染shRNA到心肌细胞是关键。本项目组前期利用超声靶向微泡破坏介导shRNA基因转染血管内皮细胞可促血管生长有效治疗心肌缺血；然而微泡不能透过血管壁，难以转染心肌细胞。. 基于以上理论和基础,本项目拟制备超声纳泡介导Gal-3shRNA转染抑制心肌纤维化和阻止LVR的效果，主要研究内容包括：(1)制备高包载Gal-3shRNA的纳泡；(2)体外实验检测不同参数下超声靶向纳泡破坏介导Gal-3shRNA的转染效率及沉默效应；(3)制备LVR大鼠模型，评价超声靶向纳泡破坏介导Gal-3shRNA转染心肌细胞抗LVR的作用，并研究其机制，本项目实施可为心衰防治提供新思路。

心肌纤维化是左室重构和慢性心功能障碍的主要病理基础，是慢性心力衰竭的主要原因。半乳糖凝集素-3（Gal-3）是多种器官纤维化的共同因素，尤其在心肌纤维化过程中发挥重要作用。虽然抑制Gal-3表达已成为一种减少纤维化和预防左室重构的新策略，但如何有效抑制Gal-3在心脏的表达仍是临床转化过程中的关键问题。短发夹核糖核酸（shRNA）是近年来发展起来的基因治疗新方法，能够显著沉默靶基因的表达，并且其抑制效果能够维持数周至数月。因此，本项目拟构建Gal-3 shRNA沉默Gal-3表达，从而抑制心肌纤维化阻止左室重构。然而如何实现Gal-3 shRNA靶向转染，是制约其发展的关键因素之一。超声靶向微泡破坏技术 （UTMD） 是一种理想的药物或基因转运载方法，可以利用微泡破坏产生的空化效应促进体内转染，实现无创靶向递送核酸。本研究拟构建载Gal-3 shRNA微泡，联合UTMD沉默Gal-3基因，降低心肌组织中Gal-3合成，从而抑制纤维化，保护心功能。为了验证假设，我们通过薄膜水化法制备阳离子微泡，通过电荷-电荷相互作用吸附Gal-3 shRNA。并在大鼠心肌梗死模型中应用UTMD技术实现Gal-3 shRNA心脏靶向转染，通过超声心动图检测大鼠左心室射血分数（LVEF）, Massson染色观察心肌纤维化，PCR及Western blot检测心肌组织Gal-3、I型胶原和III型胶原表达情况。结果显示：①成功制备了Gal-3 shRNA/CMBs，其性质稳定且生物安全性良好。②Gal-3 shRNA/CMB + US组心肌组织中Gal-3表达降低，心肌纤维化面积下降，胶原合成减少，而LVEF均高于其他各组。因此，我们认为UTMD介导Gal-3 shRNA体内转染能够有效沉默心肌组织中Gal-3表达，降低心肌纤维化程度，保护心功能，为心衰防治提供新思路。