基因枪转染SDF-1α基因自动捕获内皮祖细胞预防显微血管吻合后血栓形成

Anastomotic thrombosis is a devastating complication in microsurgery that may cause significant morbidity to patients. Despite of antithrombotic drug therapy, microsurgical procedures still carry a significant risk of thrombosis. Actually, the injured endothelial at anastomosis site resulted in the denuded subendothelial layer collagen, which aroused the cascade process of thrombosis formation. Endothelial progenitor cells (EPC) from bone marrow have been shown to play an important role in repair and re-endothelialization following vascular injury. One critical interaction in mobilization and homing of EPC is between the G-protein-coupled receptor CXCR4 and its ligand, stromal cell-derived factor-1 alpha (SDF-1α). We hypothesize that spontaneously capturing the circulating EPCs based SDF-1α/CXCR4 axis at anstomosis site will efficiently prevent from thrombus formation. The SDF-1α gene is transfected into anastomotic vascular intima using the gene gun, therefore, anastomotic intima tissue will express SDF-1α. Increasing SDF-1α secretion by gene gun transfection of SDF-1 gene would induce EPC mobilization from bone marrow into the peripheral blood and homing in anastomotic site, the process was mediated by SDF-1α/CXCR4 axis. The homing EPC will quickly reendothelialize the anastomotic intima and prevent from the thrombosis formation.

小血管吻合口血栓形成的主要原因是血管内皮的损伤和缺失, 因此, 加速吻合口的再内皮化、尽快修复损伤的血管内皮是解决问题的关键，以往采用全身抗凝或局部基因转染表达溶栓酶的方法治标不治本。最新研究表明，内皮祖细胞是修复缺损内皮的主要参与者。本项目设想，通过使小血管吻合口自动捕获外周血中内皮祖细胞这一新策略，防止吻合口血栓形成。采用基因枪对小血管吻合口内膜定向转染基质细胞衍生因子-1α（stromal cell-derived factor-1α,SDF-1α）基因，使吻合口表达SDF-1α。已知SDF-1α是内皮祖细胞的强大趋化剂，它可迅速动员骨髓基质中内皮祖细胞进入外周血中, 并通过与其表面的特异性受体CXCR4结合,自动捕获外周血中的内皮祖细胞，使其迁移、归巢到吻合口，从而加速吻合口再内皮化、修复损伤的内皮，为防止小血管吻合口血栓形成提供一种有前景的新方法。

血管损伤及随之发生的血栓形成和损伤血管病理修复过程是导致显微外科血管吻合术后失败及长期预后不佳的关键因素。本项目针对以上问题展开，主要包括以下内容：以大鼠血管吻合模型为研究对象，应用通畅实验评估血管吻合口通畅情况，采用组织学及扫描电镜技术检查血管吻合口血栓形成及损伤血管修复情况；以大鼠股动脉内膜损伤后吻合模型为对象，通过局部转染基质细胞衍生因子动员内源性内皮祖细胞，并检测其对吻合口血栓形成的影响，应用免疫荧光技术检测血管吻合术后吻合口内皮祖细胞募集情况；建立大鼠颈总动脉吻合模型，使用AMD3100阻断SDF-1/CXCR4信号轴，评估该信号轴在血管吻合术后内皮祖细胞募集及血管修复中的作用；同时将内皮祖细胞的应用拓展到缺血性皮瓣转移术后血管再生过程中，检测改善内皮祖细胞功能能否提高糖尿病大鼠皮瓣转移术后内皮祖细胞募集、血管再生及皮瓣存活率。通过上述研究可以得到以下结论：（1）显微血管吻合术导致吻合口周围内皮细胞损伤脱落，短期内诱发血栓形成，长期则可引起内膜增生；（2）基质细胞衍生因子-1α可能通过刺激一氧化氮合成释放而抑制吻合口血栓形成；（3）内源性内皮祖细胞可能通过SDF-1/CXCR4信号轴参与吻合口损伤血管修复，抑制吻合口内膜增生；（4）改善内源性内皮祖细胞动员及功能可能通过促进血管再生而改善糖尿病大鼠缺血性皮瓣的存活率。