mAb2G4引导NF-κB诱骗性寡核苷酸对缺血心肌靶向保护作用的实验研究

The myocardial ischemia reperfusion injury (MIRI) is a main interference in the reperfusion therapy for acute myocardial infarction. The treatment of myocardial ischemia using gene therapy is a rather novel but promising approach.Recently, investigations have demonstrated that anti-myosin monoclonal antibody 2G4 (mAb2G4) specific toward cardiac myosin for targeted gene delivery to ischemic myocardium.In our study we also found that NF-κB decoy ODN could reduce myocardial reperfusion injury in rats.So we intend to establish the neonatal SD rats' myocardial cell hypoxia-reoxygenation model and myocardial ischemia reperfusion injure model by ligaturing anterior descending branch of left coronary artery for 30 minutes and perfusing for 120 minutes in rats.Genetic engineering technique was applied to construct a new kind of liposome complex: anti-myosin mAb2G4/ NF-κB decoy ODN / liposome complex( mAb2G4/ODN/lip). We will investigate by different treatment pathways and treatment times whether NF-κB decoy ODN could be targeted introduced into injured heart cells in rats beacause that mAb2G4 has specific binding to ischemic myocyte. And we will also explore whether mAb2G4/ODN/lip could reduce the MIRI by inhibiting the transcription of NF-κB. We believe that our research achievements can provide theoretical and practical basis to targeted therapy for MIRI.

心肌缺血再灌注损伤（MIRI）是阻碍缺血心肌从再灌注疗法中获得最佳疗效的主要难题。近年有研究报道抗肌球蛋白单克隆抗体(anti-myosin monoclonal antibody 2G4， mAb2G4)能高效特异性地与受损心肌细胞相结合；我们前期研究发现，核转录因子-κB诱骗性寡核苷酸(NF-κB decoy ODN)可减轻MIRI。因此，我们设想建立大鼠体外心肌细胞缺氧复氧模型和在体心肌缺血再灌注损伤模型，运用基因工程技术将mAb2G4和NF-κB decoy ODN融合成脂质体复合物（mAb2G4/ODN/lip），通过不同处理时间、处理途径的比较分析来探讨mAb2G4/ODN/lip是否能利用mAb2G4与缺血心肌细胞特异性相结合的特性，将NF-κB decoy ODN靶向导入受损心肌，通过抑制NF-κB诱导的炎性反应来保护心肌；为临床靶向治疗MIRI提供一定的理论和实践依据。

心肌缺血再灌注损伤，已成为阻碍缺血心肌从再灌注疗法中获得最佳疗效的主要难题。本课题在前期研究的基础上，通过结扎大鼠冠状动脉左前降支30分钟后再灌注2小时建立大鼠在体心肌缺血再灌注模型，进一步证实以脂质体为载体的NF-κB寡聚脱氧核苷酸（ODN/lip）可抑制NF-κB的表达，降低炎性因子TNF-α、IL-6以及LDH、MDA的水平，减轻心肌细胞损伤程度，改善心脏功能，其机制可能与抑制NF-κB介导的炎性反应有关。由于mAb2G4可高效特异地识别受损心肌细胞释放出的心肌肌球蛋白片段，使NF-κB 寡聚脱氧核苷酸准确识别受损心肌，并在受损心肌特异性的表达。因此经过mAb2G4修饰的ODN/lip，即mAb2G4/ODN/lip，相较ODN/lip而言，更有效的抑制NF-κB活性，降低TNF-α、IL-6、LDH和MDA水平，减缓心肌损伤。此外，同高剂量的ODN/lip、mAb2G4/ODN/lip相比，低剂量组均显示出更好的心肌保护作用，其机制可能与过量的阳离子脂质体与细胞膜发生非特异性反应，引起胞膜溶解，细胞坏死有关。同时，体外细胞实验结果同样证实，低剂量的的mAb2G4/ODN/lip比高剂量的mAb2G4/ODN/lip更能保护心肌细胞。此次实验结果为临床治疗心肌缺血再灌注损伤提供了有利的依据。