靶向干预NF-kB信号通路防治老龄缺血性心力衰竭的作用机制研究

Aging is considered as a major risk factor for ischemia heart failure , reverse the pathological and physiological cardiac remodeling process is the key factor to control the development of congestive heart failure. Recently, we have identified that the excessive expression of MMP-9 and type Ⅲ collagen and enhancement of inflammatory cells infiltrating play an important role in cardiac remodeling of the aged heart, meanwhile NF-κB signal pathway was activated in aging chronic cardiac remodeling process, but the mechanisms are not clear. Previous studies have demonstrated that inhibited of NF-κB activation results in attenuated younger mouse cardiac remodeling and improved heart function. So, this research major in exploring the effects and mechanisms of NF-κB signal pathway in ischemic congestive heart failure of aged mice, meanwhile select the NF-κB signal pathway as intervention target, construct the 9 serotype recombinant adeno-associated viral vectors containing P65 ribozyme gene and target transfected into the heart , investigate the index of NF-κB signal pathway activity and related protein, collagen protein and inflammatory factors expression, MMPs activity, myocardial pathological damage, infarct size and cardic function, et al. Meanwhile investigate the targeted inhibition of NF-κB signaling pathway to prevent and cure the aging ischemic heart failure effect and molecular mechanisms, to identify gene targeting for therapeutic intervention effectiveness, then provide a new therapeutic strategy and theory support to gene therapy of heart failure in aged patient.

老龄是缺血性心力衰竭发病的高危因素，逆转心室重塑的病理生理过程是控制心衰发生的关键。我们前期研究发现MMP-9及III型胶原的过度表达和炎性细胞浸润增强是导致老龄缺血性心室重塑的重要原因，且NF-κB信号通路在心室重塑过程中持续激活，但具体机制不详。研究表明抑制NF-κB通路的激活能减轻年轻小鼠心室重塑、改善心功能。据此，本课题拟采用老龄小鼠缺血性心衰模型，探讨NF-κB通路在老龄缺血性心衰中的作用及分子机制；同时，以NF-κB信号通路为干预靶点，利用抗NF-κB P65核酶基因的重组AAV-9载体，靶向转导老龄小鼠心脏，通过检测NF-κB活性及通路中相关蛋白、炎症因子及胶原表达、细胞凋亡、MMPs活性、心肌病理损害程度、心功能等指标，阐明靶向抑制NF-κB信号通路防治老龄缺血性心力衰竭的作用及分子机制，明确基因靶向干预治疗的有效性和可行性，为老龄缺血性心力衰竭的基因治疗提供新的思路和依据

老龄是缺血性心力衰竭发病的高危因素，逆转心室重塑的病理生过程是控制心衰发生的关键，目前尚无根治心衰的药物和疗法。本研究探索了IκBα基因靶向转导防治老龄缺血性心力衰竭的可行性及其机制。针对目前心脏疾病基因治疗载体存在心肌的靶向性差、转染效率低、持续时间短、安全性差等缺点，利用杆状病毒载体系统，分别构建重组CMV启动子的单链及双链AAV9病毒载体。研究发现dsAAV9-CMV载体可介导外源基因在体内外高效快速表达，尾静脉转导5周在心肌转染效率可达88.6%，并可长期稳定表达至8周，且CMV启动子在肝脏中很容易被沉默，对心肌具备高效转染、靶向、长期稳定表达。且dsAAV9和ssAAV9载体对心肌酶、肝功及肾功均无不良影响。抑制NF-κB通路的激活能减轻年轻小鼠心室重塑、改善心功能，据此本课题设计并构建了表达IκBα基因的重组dsAAV9载体，dsAAV9-IκBa转导可有效抑制老龄梗死心肌P65和P50亚基的核转位、抑制NF-κB信号通路活性，IκBa基因过表达具有抗击心肌细胞凋亡，抑制心肌慢性心室重构，减少心肌梗死面积，降低缺血性心脏破裂和心力衰竭的死亡率、改善老龄小鼠的心功能和生存率的功能，从而为临床防治老龄缺血性心力衰竭、改善预后提供新的思路和干预靶点。