Annexin-V-SDF-1融合蛋白促进梗死心脏修复的机制研究

The beneficial effects of stromal cell-derived factor-1 (SDF-1), in terms of promoting myocardial repair, are limited by its low concentration in the infracted heart. Though supplementation of exogenous SDF-1 was shown to augment local SDF-1 concentration and facilitate myocardial repair, there are several important limitations with the current delivery approaches. We found in our previous study that a novel bioengineered fusion protein, Annexin-V-SDF-1, was associated with greater functional improvement of the infarcted heart in comparison with SDF-1. However, the underlying mechanism is to be studied. Our in-vitro experiments indicated that Annexin-V-SDF-1 acquired the ability of targeting apoptotic cells while retained the biological activity of SDF-1. Additionally, apoptotic cells are present at the infarct and border zone through the whole period of myocardial repair. Therefore, we hypothesize that Annexin-V-SDF-1 could specifically bind to apoptotic cells and accumulate in the infarcted heart, which in turn boost angiogenesis and cardiomyocyte regeneration via homing of stem cells. In current study, we aim at confirming the high efficiency of Annexin-V-SDF-1 in promoting myocardial repair and probing into the underlying mechanism. This will highlight theoretical basis for comprehensively understanding the biological effects of SDF-1, as well as provide a new approach to optimizing post-infarction myocardial repair.

局部低浓度将显著降低基质细胞衍生因子（SDF-1）促进梗死心脏修复的生物学功效，补充外源性SDF-1可以提高局部浓度、促进心脏修复，但现有方法存在较多局限。我们初步研究显示，与单纯的SDF-1相比，新型融合蛋白Annexin-V-SDF-1的补充更明显地改善了梗死心脏的功能，但确切机制尚不清楚。体外实验发现该融合蛋白可靶向性地结合凋亡细胞，同时保留SDF-1的生物学功效，鉴于凋亡细胞较长时间、广泛地存在于心肌梗死及周边区域，我们推测Annexin-V-SDF-1可靶向结合凋亡细胞，在梗死区域高浓度聚集，从而促进相关修复细胞归巢，加速血管新生和心肌再生等修复过程。本项目针对小鼠心肌梗死模型，进一步验证Annexin-V-SDF-1促进梗死心脏修复的高效性，并深入探讨该融合蛋白促进梗死心脏修复的机制，为全面阐明SDF-1的生物学功效和寻找修复梗死心脏的优化措施提供重要的理论依据和新的研究思路。

已有研究显示心肌梗死后SDF-1在损伤心肌局部表达时间与其受体CXCR4表达时间上的错位可能导致了SDF-1功效的下降。故人们想到外源性给予SDF-1，使SDF-1-CXCR4轴在心梗后被有效激活。而目前的给药途径难以使外源性SDF-1高效地到达损伤心肌局部。因重组蛋白Annexin V可以靶向聚集于损伤心肌局部，现已被用作为核素心肌显像中死亡心肌的示踪剂。该项目旨在将SDF-1与Annexin V融合为SDF-1-Annexin V重组蛋白，以期得到一种既能靶向聚集于梗死心肌局部又具有SDF-1功效的融合蛋白。我们首先采用重叠PCR的方法将SDF-1及Annexin V的基因编码序列连接并克隆到pET28a载体上，以构建重组表达质粒。然后，我们通过BL21（DE3）感受态大量表达SDF-1-Annexin V融合蛋白包涵体，通过on-column蛋白复性及纯化技术，我们成功纯化出较高纯度的可溶性SDF-1-Annexin V融合蛋白。在体外实验中，我们分别验证了双功能融合蛋白SDF-1-Annexin V的生物学效应，包括SDF-1结构域对CXCR4的结合能力、下游信号通路的激活，趋化作用及新生血管作用，以及Annexin V结构域对凋亡细胞的特异性识别及结合。体内实验中，我们验证了SDF-1-Annexin V对损伤心肌的靶向聚集作用，对梗死心肌新生血管形成的促进作用，尽管如此，我们发现SDF-1-Annexin V融合蛋白并没有改善心梗小鼠心功能，反而，可能加重了心梗小鼠的心肌病理性重构。本实验的结果提示：1. SDF-1与Annexin V串联融合的方式所产生的融合蛋白仍分别具有SDF-1及Annexin V蛋白的相应生物学功能； 2.Annexin V可以作为一种蛋白载体，将另一种功能蛋白运输到损伤心肌局部发挥作用，这一发现有助于开创一条药物靶向修复梗死心肌的治疗途径；3. 一味地增加梗死心肌局部SDF-1的量或延长SDF-1在梗死心肌局部作用时间并不能有效修复梗死心肌，该结果为SDF-1在临床上的正确应用提供了重要参考。