Krt5和P63双阳性气道干细胞构建组织工程气管可行性研究

The purpose of this project is to test the potential function of trachea basal stem cell TBSC (Krt5 and P63 double positive) in the reconstruction of tissue engineered trachea. Former studies have proved the function of TBSC in the regeneration of lung tissue. Due to the fact that TBSC original from trachea epithelia basal tissue, we hypothesis that it might involve and accelerate the re-epithelialization process of tissue engineered trachea. in our former NSFC project, In-vivo bioreactor for tissue engineered trachea reconstruction, we successfully repaired 8cm long trachea defect with tissue engineered trachea. Follow the theory of "in-vivo bioreactor", we performed the first tissue engineered trachea clinic application in Asia. In this project, we plan to use sheep animal model, harvest autologous TBSC through bronchoscopy, amplify in vitro and marker the cells with green fluorescent protein GFP. Part of the trachea, 8cm long, was then resected and reconstruct with tissue engineered trachea following the principle of "in-vivo bioreactor". In the "in-vivo bioreactor" design, we insert two catheters inside implanted tissue engineered trachea. The two ends of the catheters were connected to two portable perfusion pumps respectively. One pump administrate medium in while the other sucking the waste out. Through this design we create a continuous medium perfusion inside the implanted tissue engineered trachea substitute. The medium perfusion will support the survive of seed cells. We also re-seed stem cells onto the implanted tissue engineered trachea through the perfusion tubes. The TBSC with GFP , 10*7 in average amount, was then seeded onto implanted tissue engineered trachea through in-vivo bioreactor twice per week for three months. After the complete of re-epithelialization process of tissue engineered trachea, which normally takes three months, we harvest the sample and identify the marked TBSC in epithelial tissue through fluorescence microscope. if we could prove the function of TBSC in the re-epithelialization process of tissue engineered trachea. A combination of TBSC and bone marrow stem cells in the reconstruction of tissue engineered trachea will accelerate the re-angiogenesis and re-epitheliazation process of tissue engineered trachea and benefit the clinic application

本课题目标是验证Krt5和P63双阳性气道干细胞（trachea basal stem cell TBSC）构建组织工程化气管上皮组织可行性。前期动物实验模型和临床预实验证实了TBSC细胞参与了肺损伤的修复，鉴于其来源于气道基底膜，我们合理推测TBSC或将参与并加速组织工程化气管上皮重建。课题组2017年结题的国自然面上项目，“体内生物反应器构建组织工程化气管”，成功构建了组织工程化气管动物实验模型，在此基础上课题组完成了亚洲首例组织工程化气管临床实验。本课题中，我们利用羊为实验动物，通过气管镜获取TBSC，体外扩增并通过逆转录病毒标记绿色萤光蛋白，同时构建组织工程化气管修复实验羊8公分气管缺损，通过“体内生物反应器”反复接种经过标记的TBSC。三个月后取出植入的组织工程化气管，取材上皮组织，萤光显微镜验证是否存在接种的TBSC，验证其参与组织工程化气管上皮重建的假说。

前期动物实验模型和临床预实验证实了TBSC细胞参与了肺损伤的修复，鉴于其来源于气道基底膜，我们合理推测TBSC或将参与并加速组织工程化气管上皮重建。本课题中，我们以羊为实验动物，通过气管镜获取TBSC，体外扩增并通过逆转录病毒标记绿色萤光蛋白，观测其在组织工程化气管再血管化和再上皮化过程中的协调促进作用。另外，课题组成功开展临床实验，进一步探究“体内生物反应器”构建组织工程气管进行临床应用的可行性。.动物实验证实，TBSB细胞促进了组织工程化气管上皮重建，在术后2月即形成了完整的上皮，3个月的病理切片结果均显示了TBSC可通过“体内生物反应器”促进组织工程气管的再生，实验羊最高存活1.5年，因实验需要，在术后1.5年牺牲。同时在研期间，课题组获得了两项专利：“组织培养用监测辅助系统及其辅助控制方法”，“一种用于检测组织工程疗效的方法及其装置”。同时实现了“体内生物反应器”的成果转化，将“体内生物反应器”构建组织工程化气管，作为临床新技术，应用于临床。