CREKA-纤维蛋白靶向体系介导干细胞归巢治疗心肌损伤的实验研究

Poor cell homing is one of the major obstacles for cardiac cellular therapy to achieve an ideal functional benefit. In our previous studies supported by National Natural Science Foundation of China, we realized that the precise cell homing to the injured myocardium depends on the biological homing mechanism, while the external magnetic field can only promote the transient and imprecise stay in the heart. Actually, it has long been a hot issue to find a novel and effective homing target in cardiac cellular therapy. Fibrin, a transitional matrix component after tissue injury, has pleiotropic effects on issue repair, including mediating the migration of inflammatory cells and the proliferation of stem cells. Interestingly, we found that the location of the fibrin after myocardial ischemia/reperfusion was consistent with the site of myocardial injury, and the duration of the fibrin was coincident with the optimal timing of cell transplantation. We also demonstrated the homing peptide l-CREKA (l-type peptide cysteine-arginine-glutamic acid-lysine-alanine) could specifically bind to fibrin molecules in the injured heart, rather than the fibronogen in the blood. Thus, it is logical to hypothesize that fibrin may be a potential and new target in the stem cell therapy for the myocardial infarction, and the "stem cells-CREKA-fibrin" targeting system may recruit the exogenous stem cells to the injured and fibrin-rich heart. We demonstrated that d-type peptide CREKA(d-CREKA) is more stable than l-CREKA in serum. In this project, we will firstly connect the phosphorylated d-CREKA to the membrane of mesenchymal stem cells by using the liposome membrane fusion technology and the fluidity of the lipid bilayer. In in vitro study, we will evaluate the affinity of fibrin to the d-CREKA-modified stem cells, the stability of the connection between d-CREKA and stem cells, and the potential cytotoxicity of d-CREKA insertion into cell membrane. In animal study, the d-CREKA modified mesenchymal stem cells are to be injected into the left ventricle or the tail vein of the myocardial ischemia-reperfusion model of rats, and the transplanted cells will direct to the fibrin in the injured region to improve cardiac performance. Our study offers a brand-new target for transplanted cells homing to the injured myocardium, and provides new perspectives for the biologically targeted therapy of the injury of other tissues.

细胞归巢效率低下严重制约干细胞移植治疗缺血性心脏病的疗效。尽管外加磁力能促进干细胞心脏停留，但精准的组织学归巢仍有赖于生物性机制，寻找归巢新靶点是近年研究热点。纤维蛋白（FB）是心肌损伤的过渡性基质成分，具有促进细胞迁移和增殖等作用。新近我们发现FB表达部位与心肌缺血损伤部位一致，表达时间与细胞移植时机契合，由此推测FB是细胞靶向治疗心肌损伤的新靶点。项目组证实噬菌体展示技术筛选出的归巢肽CREKA能特异性靶向心肌损伤部位的FB,并构建“CREKA-FB”靶向体系。本项目拟采用脂质体融合技术和脂质流动效应，将更加稳定的右旋肽d-CREKA修饰在干细胞表面；在体外评价d-CREKA修饰干细胞的稳定性、FB靶向性和安全性的基础上，将其经血管移植到大鼠心肌损伤模型，观察细胞的心脏归巢效率和移植疗效。本研究旨在挖掘促进干细胞心脏归巢的新靶点，并为其他组织损伤的生物靶向治疗提供新视角。

细胞归巢效率低下严重制约干细胞移植治疗缺血性心脏病的疗效，寻找归巢新靶点是近年研究热点。纤维蛋白（FB）是心肌损伤的过渡性基质成分，我们发现FB表达部位与心肌缺血损伤部位一致，表达时间与细胞移植时机契合，推测FB是靶向治疗心肌损伤的新靶点。本项目中，我们利用CREKA-FB体系构建一个新型的靶向递药平台，针对心肌损伤区细胞外基质纤维蛋白靶向递送间充质干细胞（MSC），优化心肌修复疗效。.①CREKA-MSC的构建：合成、质谱鉴定归巢肽CREKA后，将其与磷脂、荧光卵磷脂等构建脂质体（CREKA-Lipo），通过膜融合技术与脂质层流动效应构建CREKA修饰MSC（CREKA-MSC）。②CREKA-MSC的体外靶向性：静态条件下CREKA-MSC对FB靶向性增加2.6倍，平行板流动条件下CREKA-MSC对FB的靶向粘附性增加2.3倍。小室趋触性迁移实验进一步证实CREKA修饰增强MSC靶向FB的迁移能力。③CREKA-MSC-FB靶向体系的生物学效应：检测CREKA-Lipo与MSC融合后不影响MSC的活力、细胞表型、分化、免疫调节功能。④CREKA-MSC靶向治疗心肌损伤：建立大鼠I/R心肌损伤模型，经左室腔移植CREKA-MSC，通过分子影像学和组织学检测细胞的靶向归巢和全身分布情况，发现CREKA修饰后，梗死区MSC细胞数量显著增加。细胞归巢的增加最终转归为细胞移植疗效的改善，机制可能与新生血管、免疫调节作用有关。⑤在上述实验基础上，我们自主研发稳定性更好的d-CREKA归巢肽，证实右旋体d-CREKA对纤维蛋白的体内外靶向性均优于左旋体l-CREKA。.本项目提出以纤维蛋白（FB）为细胞归巢靶点，是独立于外加磁场和传统细胞因子的新兴靶点，是对心肌损伤细胞靶向策略的创新性尝试。更重要的是，由于FB形成是机体任何组织损伤后的一种共同性病理反应，因此“CREKA-FB靶向体系也有望应用于各种组织损伤的生物靶向治疗（包括细胞因子靶向、药物靶向等）。