摘要
目的建立一套简单、高效、稳定、规范的人脐带间充质干细胞体外制备与鉴定技术,为人脐带间充质干细胞库建设及临床应用提供技术规范。方法改进前期技术方法,重复验证,建立规范化技术方法。无菌采集足月剖宫产分娩胎儿脐带,采用不剥离血管和包膜组织贴壁法培养脐带间充质干细胞。待原代细胞长出时,收集原本换液应丢弃的组织块,进行二次贴壁培养,如此反复,待二次贴壁组织块长出细胞时,收集组织块进行三次贴壁培养。对三次贴壁培养长出的第三代细胞进行生物学特性分析。结果首次组织贴壁后10～12 d可见成纤维样细胞从组织块爬出,二次、三次贴壁后第2 d就可见成纤维细胞样细胞爬出,且生长状况好,细胞形态更为均一。三次贴壁培养的第三代细胞都高表达CD29、CD44、CD90和CD105,不表达CD34和CD45。细胞生长曲线呈＂S＂型,均具有成脂、成骨、成软骨分化的能力。结论采用多次贴壁法制备的人脐带间充质干细胞充分利用了脐带组织块,且得到的原代细胞数量翻倍,其生物学特性稳定。
Objectivc To establish a simple, efficient, stable, and standardized human umbilical cord mesenchymal stem cell preparation and identification technology in vitro, and to provide technological specification for the establishment and clinical application of human umbilical cord mesenchymal stem cell bank. Methods The technique and method in the early time were improved and repeatedly verified. A standardized technical method was established. The umbilical cords of fetuses taking cesarean delivery at full term were aseptically collected. The umbilical cord mesenchymal stem cells were cultured by the method of not stripping the vessel and coated vascular tissue adherent method. When the primary cells grew, the tissue blocks which should be discarded at first were collected and adherently cultured for the second time. Repeatedly like that, the tissue blocks were collected for the third time adherent culture until the cells grew out in the blocks cultured at the second time. The cells of the third generation cultured during the three times of adherent culture were analyzed for the biological characteristics, Results Fibroblast-like cells could be seen climbing out 10-12 d after the first time adherence and on the third day after the second and third time of adherence. The growing condition was good, and the cell shape was more uniform. CD29, CD44, CD90, and CD105 were all highly expressed in the cells of the third generation cultured during the three times of adherent culture. However, CD34 and CD45 were not expressed. The cell growth curve was S type, and the cells could be induced to differentiate into adipocytes, osteogenesis, and chondrocytes. Conclusion The preparation of human umbilical cord mesenchymal stem cells by the method of multiple adherence method fully takes advantage of the umbilical cord tissue blocks and gets the number of primary cells doubled, and its biological characteristics is stable.
出处
《西南国防医药》
CAS
2015年第8期828-831,共4页
Medical Journal of National Defending Forces in Southwest China
基金
国家科技支撑计划(2014BI01B0)
云南省战略性新兴产业专项(2013DA004)
云南省科技创新平台建设项目(2013DA004)
关键词
脐带间充质干细胞
分离
贴壁培养
umbilical cord mesenchymal stem cell
isolation
adherent culture
