PPENK-MIDGE-NLS转染白细胞减轻心肌缺血再灌注损伤及机制

Myocardial ischemic preconditioning/postconditioning are potent endogenous protective mechanisms against ischemia-reperfusion injury. Although enkephalin can mimics preconditioning/postconditioning induce significant myocardial protective effects, but the clinical application of opioids have many limitations at ischemic circumstances. It has been demonstrated that leukocyte can produce and secrete endogenous opioid peptide, but without specificity and the amount was also not abundant. The purpose of this study is to construct PPENK-MIDGE-NLS gene vector of enkephalin by minimalistic immunologically defined gene expression (MIDGE) and then intravenously inject PPENK-MIDGE-NLS to transfect leukocyte. Combining the advantages of leukocytic aggregation and infiltration to the myocardial injury site, PPENK-MIDGE-NLS induces leukocyte continues pruduce and targeted release endogenous enkephalin in myocardial injury area. Transfection efficiency will be confirmed by flow cytometry. The effects of PPENK-MIDGE-NLS on gene expression and content of enkephalin precursor protein proenkephalin(PENK), ENK and metabolites are detected by RT-PCR, Western Blot, immunohistochemistry and radioimmunity at myocardial tissue. The feasibility and efficacy of PPENK-MIDGE-NLS gene vector inducing leukocyte to increase content of endougenous enkephlin for myocardial protection are also evaluated by the changes of haemodynamics, infarction area, cTnI and NF-κB. Based above experimental results, we try to prove an innovative method for preventing and treating of myocardial ischemia-reperfusion injury and investigate the regulative mechanisms of enkephlin on imflammation after myocardial ischemia.

心肌缺血预/后处理是对抗缺血再灌注损伤的强大内源性性保护机制，脑啡肽可模拟该机制产生显著心肌保护作用，但阿片类药物的临床应用受到诸多限制。体内免疫细胞虽能合成、分泌内源性阿片肽，但缺乏特异合成脑啡肽的能力而且量微。本课题采用微量免疫原定义的基因表达（MIDGE）方法构建前脑啡肽原PPENK-MIDGE-NLS基因载体，静脉注射转染白细胞，利用白细胞向缺血区聚集、浸润的特性，在损伤区域持续产生、靶向释放内源性脑啡肽。流式细胞术证实转染效果，分子生物、免疫组化和放射免疫法测定PPENK-MIDGE-NLS对心肌局部脑啡肽前体、脑啡肽及其代谢产物基因表达和含量的影响，结合心肌功能、梗死面积的测定及心肌酶谱和炎性因子的变化，评价PPENK-MIDGE-NLS基因载体减轻心肌缺血再灌注损伤的可行性、有效性及对炎性反应的调节作用，探讨心肌缺血再灌注损伤预防和治疗的新方法及机制。

心肌缺血预/后处理是对抗心肌缺血再灌注损伤的强大内源性性保护机制，脑啡肽可模拟该机制产生显著心肌保护作用，但阿片类药物的临床应用受到诸多限制。体内免疫细胞虽能合成、分泌内源性阿片肽，但缺乏特异合成脑啡肽的能力而且量微。本课题采用微量免疫原定义的基因表达（Minimalistic Immunologically defined gene expression,MIDGE）方法构建前脑啡肽原PPENK-MIDGE-NLS基因载体，静脉注射转染白细胞，利用白细胞向缺血区聚集、浸润的特性，在心肌损伤区域持续产生、靶向释放内源性脑啡肽，从而产生心肌保护作用。本课题利用分子生物学方法成功构建了PPENK-MIDGE-NLS基因载体，转染实验证实了该基因载体对体细胞、心肌细胞和白细胞的转染效果。为进一步评价PPENK-MIDGE-NLS基因载体减轻心肌缺血再灌注损伤的可行性、有效性及机制研究奠定了基础，也为探讨心肌缺血再灌注损伤预防和治疗的新方法及机制提供了前提条件。