联合UTMD构建亲核型基因转导体系提高hAng-1基因入核率促缺血心肌血管再生

Therapeutic angiogenesis provided a new treatment for refractory ischemia. Ultrasound targeted microbubble destruction(UTMD)has been proved to be a feasible exogenous gene delivery system.However,the low gene expression level is the major challenge of this technique. Basically, cytomembrane, nuclear membrane and nuclease degradation are the three major barries of exogenous gene delivery into human cells, while previous studies mostly focused on the cytomembrane entering under UTMD,and little was known about UTMD mediated nucleus entering. This study aimed to construct an nucleophilic gene deliver system which could significantly improve therapeutic gene angiogenesis by boosting UTMD mediated gene enter nuclus. The advantages of this nuclephilic gene transport complex are that could specificlly overcome the three barries of exogenous gene get into nuclus, which known as breakthrough cytomembrane,breakthrouth nuclear membrane and not be degraded by DNA enzymes in cytoplasm.The study scheme include the following four expriments.Part one,construct nuclephilic gene carrier based on ultrasound irradiation energy and nuclear localization signal peptide(NLS)，then make identification.Part two, UTMD mediated hAng-1 gene transfect into rat H9C2 cells via nucleophilic gene delivery system, the gene expression level is detected. Part three, hAng-1 gene are transfected into canian infarcted myocardium by UTMD mediated nuclephilic gene transport complex and non- nuclephilic gene transport complex. Comprare the hAng-1 gene expression in H9C2 cells and ischemic dogs, and angiogenesis effects as well. Finally , evaulate the security of UTMD mediated nuclephilic gene delivery system. This study is trying to stand on a stone different from the past to discover how to upgrade UTMD mediated gene therapy.

超声靶向微泡破坏（UTMD）介导血管新生基因治疗为难治性心肌缺血提供了新策略。然而，胞膜屏障、核酸酶降解及核膜屏障这三个原因导致了外源基因转染效率低下。既往研究仅关注了UTMD促基因入胞，而对UTMD介导基因入核及减少DNA的降解缺乏认识。本项目组前期实验发现UTMD可能促进基因入核。故本研究拟引入核定位信号联合UTMD构建新型亲核型基因转导体系，介导治疗基因入胞后再入核并且保护胞质中的DNA，通过提高入核率来提高血管新生的疗效。主要研究内容为：构建亲核型基因转载体系并鉴定；应用亲核型基因转导体系介导hAng-1基因体外转染大鼠心肌母细胞H9C2，体内转染犬心肌梗死模型，检测转染效率、血管新生效应和心功能改善情况来评价亲核型基因转导体系的可行性及应用价值，同时评价该转载体系的应用安全性。藉此研究提升UTMD作为非病毒基因转导体系在治疗心肌缺血上的潜在应用价值。

超声靶向微泡破坏（UTMD）介导血管新生基因治疗为难治性心肌缺血提供了新策略。本研究在UTMD促进基因进入细胞的基础上，引入核定位信号（NLS）构建新型亲核型基因转导体系，介导治疗基因入胞后再入核并且保护胞质中的DNA， 通过提高入核率来增加转染效率而提高血管新生的疗效。主要研究内容为：构建亲核型基因转载体系并鉴定；应用亲核型基因转导体系介导血管生成素1（hAng-1）基因体外转染细胞，体内转染犬心肌梗死模型，检测转染效率、血管新生效应和心功能改善情况来评价亲核型基因转导体系的可行性及应用价值，同时评价该转载体系的安全性。结果显示成功构建的亲核型基因转导体系中NLS/pDNA摩尔比为104：1；PEI可以作为pDNA和NLS的链接桥梁，pDNA与PEI的最佳N/P比为2：1。PEI和NLS对DNA具有浓缩、保护作用，能保护更多外源性基因不被水解而快速进入细胞核，此时亲核载体组DNA入胞率、入核率均较非亲核载体组增高，转染效率明显提高，且不影响各组细胞转染活性。在UTMD提高细胞膜通透性促进外源性DNA进入细胞浆的基础上，经典核定位信号肽（cNLS）可促使DNA进入细胞核，可以提高体外细胞实验和体内动物实验整体转染效率，而突变核定位信号肽（mNLS）和入核阻滞剂（WGA）均会影响转染效率。动物实验中转染后，标记蛋白EGFP、hAng-1、心肌新生血管密度、实时心肌超声造影观察心肌血管活体灌注状况均在cNLS组增加，收缩功能相对改善，梗死面积相对减少。在常规经静脉注射的基础上探索了经冠状动脉注射的方式也同样证实了带NLS的亲核型载体组联合超声辐照能提高hAng-1基因转染效率促进犬急性心肌梗死后血管新生，肌钙蛋白I（cTnI）及NT-proBNP在各组间未见显著差异，心肌细胞肌小节形态和排列在透射电镜下亲核型载体组改善更明显。本研究证实联合UTMD构建亲核型基因转导体系能提高hAng-1基因入核促缺血心肌血管再生，可作为非病毒基因转导体系在治疗心肌缺血上具有潜在应用价值。