基于孔隙可控性真空冻干脱细胞猪主动脉构建新型组织工程血管的实验研究

Because of clinical lacking of appropriate vascular graft to repair the vascular defects, serious impacts on the therapeutic effect some congenital heart diseases and vascular diseases occurred.Vascular tissue engineering is expected to become an effective means to repair these vascular defects.The first thing for construction of tissue engineered blood vessel is to find the ideal scaffold material. Decellurarized vascular conduit could be an ideal stent for its natural tissue composition and structure, good biocompatibility and biomechanical strength. However, two major problems including incomplete decellularization and the difficulty for seed cells inoculation hindered the application of decellurarized cascular conduit. Increase vascular permeability and porosity may be the key to solve the above problems.Our previous study showed that vascular permeability and porosity could be increased by the use of vacuum freeze-drying technology through controlling the speed of different pre-freezing. Based on the increase of vascular permeability and porosity, this subject will further use the siphon effect during rehydration of the freeze-dried vascular and use detergent in rehydration to improve the efficiency of decellularization in vascular membrane; and use seed cell suspension for rehydration to inhale seed cells into blood vessels so as to solve these two problems.We will use the above method to construct tissue engineered blood vessels in vitro, and to implant it into animals to observe angiogenesis situation on a regular basis.The study, if successful, will lay a good foundation for the clinical application of decellurarized vascular conduit.

先天性心脏病及其他血管性疾病日益高发，临床缺乏合适的血管移植物一直制约着这类疾病的治疗效果。组织工程血管有望为修复血管缺损提供有效的手段，而理想的支架材料是构建组织工程血管的基础。脱细胞血管具有天然的组织成份和结构，良好的生物相容性及生物力学强度而成为最为理想的支架来源。然而，天然脱细胞血管应用过程中依然存在脱细胞不完全和种子细胞接种困难两大问题。提高血管的通透性和孔隙是解决上述问题的关键。我们前期研究表明可以利用真空冷冻干燥技术，通过控制预冻速度可以提高血管的通透性和孔隙。本课题在此基础上，进一步深入探讨冻干脱细胞的机制及效果，种子细胞接种的原理及最适条件，期望同时解决脱细胞困难和种子细胞接种困难两大难题。最后拟通过间充质干细胞的接种及在体的组织工程血管实验，验证本项目采用策略的可行性，为解决临床血管移植物缺乏的难题提供实验依据。

尽管同种带瓣管道依然在临床中得到应用，但存在保存要求高、易钙化、易狭窄等缺点。本项目即探索冻干脱细胞处理对同种带瓣管道的影响，继而简化其保存条件，降低手术移植后钙化和狭窄的发生。研究中，我们以新鲜脱细胞动脉为对照，脱细胞冻干动脉为实验组，从处理大体观、组织学、机械强度等方面予以评价，发现冻干脱细胞血管没有细胞残留，同时具有良好的机械强度以满足移植条件，进而以此建立起一套完整的脱细胞冻干血管制备流程。通过猪胸主动脉移植实验，我们进一步了解到脱细胞冻干血管较新鲜冻干血管发生术后管道狭窄的可能性更低。免疫组化染色显示，冻干脱细胞血管移植物中CD3+和CD68+细胞浸润明显更少，同时移植后引起的血清TNF-α及组织中MMP-9上升更温和。因此，我们认为冻干脱细胞带瓣管道可以替代传统深低温冻存的同种带瓣管道。除此之外，我们还就同种带瓣管道缺乏的问题研究了一种新的瓣膜制备方法及一种可同时减少血小板粘附、促进内皮祖细胞粘附增殖的材料；也制备出了胶原/PLCL电纺膜，并就其在构建组织工程血管及组织工程软骨中的应用作了初步研究。上述结果将为改进小儿先心病手术材料起到重要的推进作用。