京尼平交联脱细胞同种异体气管移植的原位仿生组织工程研究

Allogenic tracheal grafts has proven its capability to repair extensive tracheal defects by using the conventional methods of tissue engineering. However, some limitations existed: (i)relatively long-lasting engineering period and high costs,(ii) potential risks of cell differentiation instability and contamination,(iii)loss of extracellular matrix architecture, decreased mechanical and angiogenic properties after in vitro long-term preservation. Bionic tissue engineering is the latest and promising approach to tracheal defect repair and regeneration. The objectives of the present study are as follows: (i) to crosslink the decellularized allogenic tracheal matrices in order to maximize its structural integrality , appropriate biomechanical property and the potential of induced neovascularization, (ii) to recellularize the cross-linked matrices (collecting and seeding intraoperatively) without using bioreactor in vitro, and to advance in situ tracheal regeneration by administering growth and regenerative factors perioperatively based on stem cells,(iii)to evaluate the function of the regenerated trachea postoperatively in an effort to tackle some key issues, such as the decreased mechanical and angiogenic properties, immunological rejection, incomplete re-epithelialization and unfavored cartilage formation and revascularization. This innovative research aims at developing a reproducible and cost-effective tracheal regeneration technique to further simplify the operation procedure, optimize the experimental process, facilitate the clinical translational application and benefit patients suffering from irreversible major airway diseases.

利用传统组织工程方法实施同种异体气管移植修复气道缺损虽有成功报道，但仍然存在以下不足：体外生物反应器处理周期长、耗费大，细胞经体外培养易出现表型不稳定和污染风险，脱细胞基质长期保存易发生结构破坏、机械强度和诱导血管生成能力的下降。仿生组织工程是气管再生领域最具探索价值的研究方向。本课题拟开展：1)对脱细胞气管基质进行交联，最大限度保持其结构的完整性、适宜的机械性能和诱导血管新生的潜能；2)基于骨髓干细胞，免除使用体外生物反应器，移植术中对经交联的脱细胞基质实施再细胞化（即时采集、即时接种），结合围术期促再生因子应用，促进气管原位再生；3）对移植术后再生气管进行功能评价。本课题力图解决再生气管的生物力学性能降低、免疫排斥、上皮化不全、软骨新生与再血管化困难等关键问题。通过创新研究极具可操作性、经济高效的原位再生技术，简化操作步骤，优化实验流程，促使基础研究成果临床转化，惠及更多气管疾病患者。

本项目主要解决的关键技术与创新点如下：.1)采用最佳浓度为5% 的NaClO4对气管基质进行脱细胞处理，在去除具有免疫原性成分的同时，保留了细胞外基质结构的完整性。将脱细胞气管基质埋入同种异体，显示经NaClO4脱细胞处理后气管基质材料的免疫原性和排斥反应显著降低。.2)采用去污剂-联合酶法（DEM）对气管基质进行脱细胞处理（7个周期），能够有效获取低免疫原性的脱细胞气管基质，该材料具有与原生气管相似的结构特征及生物力学性能。采用京尼平对脱细胞气管基质进行交联，能够有效提高脱细胞气管基质的生物力学性能，且不影响其血管生成性能，也不诱发体内炎性反应。.3)采用全骨髓贴壁培养法体外分离纯化自体骨髓间充质干细胞，并予以鉴定；基于骨髓间充质干细胞，免除使用体外生物反应器，移植术中对经交联的脱细胞基质实施再细胞化，结合促再生因子的应用，有利于气管原位再生；骨髓间充质干细胞在体内可以分化为软骨细胞，未发生明显的排斥反应。.4)绿色荧光蛋白可以在293T细胞内被包装在慢病毒内。通过梯度离心得到的自体骨髓单个核细胞可以被含有绿色荧光蛋白的慢病毒感染，并对其进行标记；通过慢病毒感染的方法对自体骨髓单个核细胞进行示踪，以鉴定移植后再生的软骨细胞是否是由术中种植的自体骨髓单个核细胞分化而来。将自体骨髓单个核细胞术中种植在经京尼平交联的脱细胞气管基质表面，术后可以分化为软骨细胞。该操作可以有效避免干细胞体外培养过程中可能带来的污染，同时也节省了时间和经济成本。