抗血管渗漏肽PEDF（44-mer）和血管生成肽PR39共表达在梗死心肌的作用及相关机制研究

Traditional gene therapy research of angiogenesis was fulfilled , but its super ability of inducing vascular permeability was often ignored,therefore,to investigate the feasibility of preventing and treating the myocardial infarction by polycistron secretory express antiangiogenesis leakage peptide and angiogenic peptide,the study will be performed by designing the recombinant AAV which could make secretory expression of the peptide PR39 - an angiogenic master switch and PEDF (44-mer), then was transduced to the coronary artery of insufficiency of hypoxic myocardial muscles by the interventional way. In the early stage of myocardial anoxia and infarction, the expression of PR39 were quickly started,and selectively inhibited the ubiquitin–proteasome-dependent degradation of hypoxia-inducible factor-1α protein (HIF-1α), resulting in the upregulation intra-cellular VEGF, KDR, FLT-1 and FGFR1, PR39 not only promoted angiogenesis,but also protected the survival，saved "the intermediate zone" in time, to prevent it from the irreversible direction，we will evaluate the effect of myocardial infarction by SSFP BOLD MR. In the late stage of myocardial infarction, AAV cloud play the antiangiogenesis leakage by expressing the 44-mer peptide and improve the angiogenesis efficiency of infarction area, we wish to clarify the molecular mechanism of the antiangiogenesis leakage peptide(44-mer).We expect to provide the new view for the prevention and treatment of cardiac and cerebrovascular disease.

传统促血管生成基因治疗多以VEGF为靶点，但忽略了其超强诱导血管渗透性的能力，往往会影响缺血区血管新生的效率。为探讨多顺反子共分泌表达抗血管渗漏肽及血管生成肽治疗心梗的可行性，以及阐明梗死区44-mer抗血管渗漏、PR39抗凋亡及促血管新生作用，本课题通过并接子技术共分泌表达“血管生成总开关”-PR39及PEDF(44-mer)重组腺相关病毒，通过介入技术将其转导至梗死区，在心梗早期启动PR39表达，选择性抑制泛素-蛋白酶体对HIF-1α的降解，来促进血管生成的细胞因子和受体（VEGF等）高表达，既促进血管新生，又抗缺氧应激凋亡，挽救更多“中间地带”缺氧细胞，行SSFP BOLD MR成像评价疗效；在梗死后期过表达44-mer，发挥其抗血管渗漏作用，提高梗死区血管再生效率，并希望阐明44-mer抗血管渗漏作用位点的分子机制研究。本课题将为完善缺血心肌促血管新生的策略，提供可靠的理论基础。

传统的AMI基因治疗大多着眼于VEGF促血管生成作用，但忽略了其过表达导致血管内皮细胞间出现缝隙，血管渗透性明显升高，从而带来副作用,往往会影响缺血区血管新生的效率。为探讨多顺反子共分泌表达抗血管渗漏肽及血管生成肽治疗心梗的可行性，以及阐明梗死区44-mer抗血管渗漏、PR39抗凋亡及促血管新生作用，本课题通过并接子技术, 成功构建了共分泌表达“血管生成总开关”-PR39及PEDF(44-mer)重组慢病毒，在低氧环境中，慢病毒可促进血管内皮细胞44-mer, 3flag-PR39,HIF-1α表达，血管渗漏相关蛋白（occludin、β- catenin、β- actin、VE-cadherin、p-VE-cadherin）表达；通过介入技术将其转导至梗死区，在心梗早期启动PR39表达，实验结果显示促进血管生成的细胞因子和受体（VEGF等）高表达，既促进血管新生，又具有抗缺氧应激凋亡作用，挽救了更多“中间地带”缺氧细胞， SSFP BOLD MR成像显示实验组心肌梗死区面积明显小于对照组；在梗死后期过表达44-mer，发挥其抗血管渗漏作用，抑制了由于VEGF过表达引起的血管内皮细胞间缝隙增宽及血管渗透性升高，提高梗死区血管再生效率，Western blot结果显示模型组 flag、occludin、P120、ZO-1 和 VE-cadherin 蛋白表达较正常降低， PR39-44mer 组蛋白表达有所上升，进一步阐明了44-mer抗血管渗漏作用位点的分子机制研究。在梗死区两个基因可同时表达，使血管生成和血管渗漏达到一个平衡，提高了血管生成效率，这是对多个生物活性短肽共表达的重要探索，为心肌梗死修复和再建提供新思维。

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