应用肝素-泊洛沙姆温敏性水凝胶提高血管吻合质量的实验研究

Vascular anastomosis plays a key role in saving life and restoring of limb function. In 1912, Alexis Carrel was awarded the Nobel Prize in Physiology and Medicine due to his contribution in improving vascular closure technology. During following 100 years, many novel techniques developed, such as medical adhesive anastomosis, cuff vascular anastomosis, built-in soluble stent method and laser welding method. However, restenosis and thrombosis after vascular anastomosis always plague the development of vascular anastomosis, especially for small blood vessels. Recently, researchers from Medicine School of Stanford university use poloxamer 407 gel combined with medical adhesive to achieve high-quality vascular anastomosis. Poloxamer is a biocompatible polymer material which has been approved using in vivo by Europe and United States. To improve the application of poloxamer in vascular anastomosis, we have synthesized a new temperature-sensitive material-heparin-Poloxamer (HP). HP not only remains the temperature-sensitive characteristics as Poloxamer, but also enjoys good anti-clotting activity as heparin. In this project, HP hydrophilic gel will be prepared and used in medical adhesive anastomosis. With the advantages of temperature-sensitive characteristics and anti-clotting activity, HP hydrophilic gel is expected to improve the shortcomings of poloxamer 407 gel and overcome the bottlenecks of anastomotic stenosis and thrombosis. With series experiments of temperature-sensitive properties, gelling temperature characteristics and vascular anastomoses in vivo, the practicality and security of HP temperature-sensitive hydrophilic gel will be fully investigated. Through the project, novel techniques will be gained to solve the bottleneck of vascular anastomosis in vascular surgery, improve surgical safety, reduce the difficulty of vascular operation, and achieve high-quality anastomosis.

血管吻合术在挽救生命和保全肢体功能的救治过程中起到关键作用。1912年Alexis Carrel 因改进血管缝合技术被授予诺贝尔生理学和医学奖。随后一百年里涌现了包括黏合法、套接法、内置支架法、激光焊接法等多项新技术，但是血管吻合术后的再狭窄和血栓形成问题始终困扰血管吻合术的发展，尤其针于微小血管。美国斯坦福大学医学院应用泊洛沙姆407凝胶结合医用黏合剂的黏合法实现血管的高质量吻合。本课题组前期自主合成了温敏型肝素-泊洛沙姆 (HP) 新材料，HP保留了泊洛沙姆的温敏性能并具有较好的抗凝血活性，将HP制成亲水凝胶，联合医用黏合剂应用于中小血管的吻合，可望改进泊洛沙姆407凝胶的不足，克服吻合口狭窄和栓塞等瓶颈问题。本课题通过温敏性能考察、胶凝温度测定和在体动物血管吻合等一系列实验，充分考察HP亲水凝胶的实用性和安全性，形成原创性成果，解决血管外科手术中血管吻合技术的瓶颈，实现高质量血管吻合

泊洛沙姆是生物安全性较好的高分子材料，以泊洛沙姆作为赋形剂的温敏性材料在多方面具有广泛应用，包括小血管和微血管吻合、药物定点释放等。虽然泊洛沙姆凝胶结合医用粘合剂的黏合方法可实现小血管及微血管的高质量吻合，但泊洛沙姆凝胶在实际应用中尚存在凝胶温度控制和抗凝等问题。本课题通过自主研发合成了温敏型肝素-泊洛沙姆（heparin-poloxamer, HP）新材料，并充分考察HP水凝胶在血管吻合技术和其它疾病治疗中应用的实用性和安全性。为解决凝胶温度的问题，本课题分别合成了共聚物HP 188与HP 407凝胶，通过比较二者物理性质和凝胶温度的区别，发现HP 407比HP 188热稳定性更好，且配制较容易，其最佳的应用浓度为35%。为进一步验证HP的安全性，本课题通过小鼠短期毒性试验证明，HP 188凝胶相较于HP 407凝胶具有一定毒性，而HP 407凝胶与正常组无显著性差异。综上结果表明，HP 407更适合应用于血管吻合术。此外，我们在HP联合药物使用中发现，神经生长因子（Nerve growth factor, NGF）复合HP 407凝胶（NGF-HP）不仅可以有效保持NGF生物活性，还能提高细胞对NGF的摄取率，并且无显著细胞毒性。进一步在大鼠脊髓损伤修复试验中发现，NGF-HP有效抑制神经胶质疤痕的形成，并促进NGF在脊髓损伤后到达神经损伤部位而达到修复神经的作用。此外，通过使用类似的办法在明胶纳米颗粒，PG脂质体系统也开展相关研究工作。本项目的完成，不仅证明了HP 407具有更好的热稳定性和生物安全性，适用于血管吻合术中的应用，还证明了其与生物活性因子的联合使用，可有效应用于组织修复，对功能性组织工程材料的设计和加工具有重要指导意义。本项目共发表文章6篇，其中SCI论文3篇，中文核心期刊3篇，并培养硕士研究生4名。