Pre-targeting/Click反应介导的自体循环干细胞在心脏缺血损伤修复中的应用及机制研究

Stem cell therapy is one of the promising strategies for the treatment of ischemic heart disease. However, the clinical application of stem cell transplantation is hampered by the immune inflammatory response and more importantly, the low stem cell retention in recipients’ infarcted myocardium. Previously, our studies have shown that platelet membrane had a targeting effect to the infarcted myocardium and the administration of bispecific antibody significantly enhanced the recruitment of cycling autologous stem cells for ischemic myocardium targeting. Therefore, the treatment outcome was improved through stem cell retention and engraftment boosting. However, the targeting efficiency of bispecific antibody was limited by steric hindrance and antigenic competition. To this end, non-cell bifunctional intelligent cell targeting system (NCBI-S) was engineered to improve stem cell therapy by fractional targeting: the first engineered antibody will pre-target onto circulating platelets that will inherently binding to the infarcted myocardium; the second one will recognize and bind to the circulating autologous stem cells in the bloodstream. Then via bio-orthogonal click reaction, the second engineered antibody will precisely anchor to the first one that already accumulated in the infarcted area. Therefore, NCBI-S will effectively bring autologous stem cells to the infarcted area for cardiac repair after ischemic injury. Based on our preliminary data, we hypothesized that NCBI-S not only strengthened myocardial regeneration and angiogenesis via stem cell paracrine effects, but also inhibited the transition from cardiac fibroblasts to myofibroblasts by suppression of bFGF/TGFβ/Smad and/or TGFβ/Erk1/2 pathways. This proposed research will test the reliability of our hypothesis and will provide new theoretical and experimental foundation for the treatment of ischemic heart disease.

干细胞应用是缺血性心脏病治疗的方法之一，但干细胞在受体心脏滞留率低和免疫炎症反应等影响其治疗效果。申请人相关前期研究发现血小板膜片有靶向效果以及用双抗体连接体捕获自体循环干细胞并靶向心梗部位可增加干细胞滞留率，治疗效果增强。但是双抗体连接体由于空间位阻、抗原竞争等原因靶向效果有限。因此，本项目在前期研究和预实验基础上设计“非细胞双功能智能靶向细胞治疗体系”（NCBI-S），首次采用分步先期靶向结合循环中血小板，利用血小板对缺血损伤区域的天然结合力，通过Pre-targeting/Click反应，将后期靶向结合的体内循环干细胞锚定在缺血损伤区域，促进心肌和血管再生，及通过bFGF/TGFβ/Smad和/或Erk1/2信号通路抑制心脏纤维化，发挥心脏缺血损伤修复的作用。通过本研究，有望证实NCBI-S的可靠性，为缺血性心脏病的治疗提供新的理论和实验基础。

急性心肌梗死 (AMI) 仍然是世界范围内死亡和发病的主要原因。严重的心肌梗死随后会导致心力衰竭。一旦心衰，剩下的唯一选择就是心脏移植。心脏细胞疗法是一种很有前景的选择，但其临床疗效一直处于边缘地位，至少部分原因是静脉内给药后细胞植入和肺中的截留率高。.我们开发了P-DSPE-PEG-CD34来捕获循环CD34阳性细胞并将它们运送到心脏损伤部位以进行有效的心脏修复。研究结果表明，CD34修饰的血小板(P-CD34)有效地捕获了CD34阳性循环干细胞并引导它们进入梗塞区域。心脏组织学表明，P-CD34治疗增强了血管肌生成和心肌细胞增殖，导致所有治疗组中存活心肌的数量最大和疤痕尺寸最小，且CD34抗体连接不会对血小板产生细胞毒性。.我们在P-DSPE-PEG-CD34的基础上设计了一个预靶向生物正交共轭(PTBC)系统，以实现无需细胞移植的内源性细胞介导的心脏修复。首先CD34抗体（与内源性干细胞结合）和CD41抗体（与血小板结合，靶向MI区域）分别使用生物正交叠氮化物和二苯并环辛炔(DBCO)连接的聚乙二醇(PEG)衍生物进行修饰。CD41附着的DBCO聚合物(DBCO-PEG-CD41)能够靶向结合血小板。将由于血小板的归巢能力，预靶向血小板积聚在MI区域。在预靶向后，给予叠氮化物修饰的CD34(Az-PEG-CD34)，它将识别并结合内源性干细胞。然后干细胞上的叠氮化物基团将识别血小板上的DBCO基团并与之反应。因此，通过生物正交点击反应，内源性干细胞与血小板结合。附着的血小板将“搭载”干细胞并将它们积聚在梗塞区域以进行心脏修复。