人骨髓间充质干细胞复合人脐静脉内皮细胞构建预血管化成骨细胞膜片  

Fabrication of prevascularized osteogenic differentiated cell sheet based on human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells

作　　者：陈佳 杨艺强 胡晨 陈琦 赵恬 雍敏[1] 马东洋 任利玲[3] Chen Jia;Yang Yiqiang;Hu Chen;Chen Qi;Zhao Tian;Yong Min;Ma Dongyang;Ren Liling(Stomatology Hospital of General Hospital,Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China;Department of Craniomaxillofacial Surgery,The 940th Hospital of Joint Logistics Support Force of Chinese PLA,Lanzhou 730052,Gansu Province,China;School of Stomatology,Lanzhou University,Lanzhou 730000,Gansu Province,China)

机构地区：[1]宁夏医科大学总医院口腔医院,宁夏回族自治区银川市750004 [2]中国人民解放军联勤保障部队第940医院口腔颌面外科,甘肃省兰州市730052 [3]兰州大学口腔医学院,甘肃省兰州市730000

出　　处：《中国组织工程研究》2020年第31期4934-4940,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81670969),项目负责人:马东洋。

摘　　要：背景:如何使工程化骨组织移植后能够成活并发挥功能,前提条件是必须含有充足有效的血液供应,而这也正是工程化组织应用于临床的主要障碍之一。目的:探索新的体外构建血管网络的方法以解决工程化骨组织预血管化的问题。方法:人骨髓间充质干细胞以9×10^4/cm^2高密度接种,经成骨诱导连续培养14 d形成细胞膜片,镜下观察细胞形貌,并进行碱性磷酸酶染色和茜素红染色。然后将培养的人脐静脉内皮细胞以5×10^4/cm^2密度接种到上述细胞膜片上,继续培养至7 d。通过镜下观察、CD31免疫荧光染色、Ⅰ型胶原免疫荧光染色评估血管网形成情况及膜片特性。结果与结论:①经连续培养可获得完整的成骨诱导细胞膜片,茜素红染色和碱性磷酸酶染色呈阳性;②倒置相差显微镜观察发现人脐静脉内皮细胞接种在成骨诱导细胞膜片上逐渐重排,CD31染色显示进行性管腔形成;③接种人脐静脉内皮细胞后构建的预血管化成骨诱导细胞膜片Ⅰ型胶原染色阳性;④结果表明,经成骨诱导后可形成具有成骨特性的细胞膜片,将人脐静脉内皮细胞培养在该膜片上可体外形成预血管化成骨细胞膜片,为优化构建预血管化的骨组织提供了新的理论指导。BACKGROUND:How to make the engineered bone tissues survive and function after transplantation,the precondition is to have sufficient and effective blood supply,which is one of the main obstacles in the clinical application of the engineered bone tissue.OBJECTIVE:To explore a new method to acquire vascular networks in vitro to realize vascularized engineered bone tissues.METHODS:Human bone marrow mesenchymal stem cells were cultured on a cell culture dish at a high density 9×10^4/cm^2 for 14 days to form a thick cell sheet.The morphology of the cells was observed under a microscope and cells were stained with alkaline phosphatase and alizarin red.The human umbilical vein endothelial cells at 5×10^4/cm^2 were seeded onto the surface of human bone marrow mesenchymal stem cells sheet for 7 days.The formation of vascular network and the characteristics of the sheet were evaluated by microscopic observation,CD31 immunofluorescence staining and type I collagen immunofluorescence staining.RESULTS AND CONCLUSION:(1)Human bone marrow mesenchymal stem cells proliferated fast and formed an intact sheet,and the sheet cultured in osteogenic-induced medium showed positive staining of alkaline phosphatase and alizarin red.(2)Inverted phase contrast microscope showed that human umbilical vein endothelial cells migrated and aligned arrangement on the osteogenic differentiated cell sheet.The immunofluorescent images for CD31 revealed a progressive process of lumen formation.(3)Positive type I collagen was observed in the pre-vascularized osteogenic differentiated cell sheet.(4)These results indicate that the osteogenic differentiated cell sheet can be formed by continuously culturing.The prevascularizing method using coculture model of culturing endothelial cells onto an osteogenic differentiated cell sheet provides a theoretical guidance for optimizing the construction of prevascularized bone tissue.

关 键 词：干细胞 骨髓间充质干细胞 预血管化 细胞膜片 血管内皮细胞 成骨 共培养 血管网 实验 

分 类 号：R496[医药卫生—康复医学] R-331[医药卫生—临床医学]