生物活性材料Col-Tgel对人组织干细胞的作用  

Effects of Col-Tgel, a bioactive material, on human tissue stem cells

作　　者：尹秀秀 胡林萍 朱才英 张孝兵 程涛 Yin Xiu-xiu;Hu Lin-ping;Zhu Cai-ying;Zhang Xiao-bing;Cheng Tao(State Key Laboratory of Experimental Hematology,Institute of Hematology and Blood Disease Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Tianjin 300020,China)

机构地区：[1]实验血液学国家重点实验室,中国医学科学院血液病医院血液学研究所,中国医学科学院干细胞医学中心,北京协和医学院干细胞与再生医学系,中国医学科学院暨北京协和医学院,天津市300020

出　　处：《中国组织工程研究》2018年第10期1540-1546,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家重点研发计划项目(2016YFA0100600);中国医学科学院医学与健康科技创新工程项目(2016-I2M-1-017);国家自然科学基金项目(81400150,81570164);创新研究群体科学基金项目(81421002);协和青年基金和中央高校基本科研业务费专项资金项目(3332016090)

摘　　要：背景:虽然传统的人干细胞二维平面培养体系操作简便,但并不能真正模拟体内的三维生理微环境,不利于进行人干细胞生物学行为研究。目的:为构建一种可为细胞生长增殖提供类似于体内生理环境的三维培养体系,观察生物材料Col-Tgel对人造血干细胞和人间充质干细胞生物活性的影响。方法:(1)体外共培养实验:三维培养组,将绿色荧光蛋白标记的人间充质干细胞与人脐带CD34^+细胞直接接触共培养于生物材料Col-Tgel中;单独培养组,将人脐血CD34^+细胞培养于生物材料Col-Tgel中;二维培养组,将绿色萤光蛋白标记的人间充质干细胞与人脐血CD34^+细胞共培养于transwell小室;对照组,常规培养人脐血CD34^+细胞。培养3 d后,流式细胞仪检测各组CD34^+CD38-CD45RA-CD90^+细胞比例,同时对直接接触培养组进行原位免疫荧光染色;(2)体内移植实验:采用X射线照射NOD/SCID小鼠,照射后24 h分2组治疗,实验组于胫骨骨髓腔移植绿色荧光蛋白标记的人间充质干细胞-生物材料Col-Tgel溶液,对照组移植绿色荧光蛋白标记的人间充质干细胞-PBS溶液,移植3 d后进行移植侧胫骨标本双光子/共聚焦显微镜检测。结果与结论:(1)体外实验:三维培养组CD34^+CD38-CD45RA-CD90^+细胞扩增倍数为二维培养组的2.8倍,说明在生物材料Col-Tgel中培养的间充质干细胞促进了CD34^+CD38-CD45RA-CD90^+细胞的扩增;三维培养组CD34^+CD38-CD45RA-CD90^+细胞扩增倍数较单独培养组增加4.5倍,较对照组增加1.5倍;免疫荧光染色显示,人间充质干细胞与人脐血CD34^+细胞可在生物材料Col-Tgel中存活;(2)体内实验显示,两组均可见存活的人间充质干细胞;(3)结果表明,生物材料Col-Tgel可为人干细胞生长及增殖提供类似于体内微环境的三维体系。BACKGROUND:The traditional two-dimensional culture system has been widely used in the in vitro culture of human tissue stem cells,but it cannot really simulate the three-dimensional physiological microenvironment in the body,which is not conducive to the study of the biological behavior of human stem cells.OBJECTIVE:To detect the effect of the bioactivity of Col-Tgel in human hematopoietic stem cells(HSCs)and mesenchymal stem cells(MSCs)in vitro and in vivo,by constructing a three-dimensional culture system stimulating the physiological microenvironment of the body.METHODS:(1)In vitro co-culture:Green fluorescent protein labeled MSCs(MSCs-GFP)and human umbilical cord blood CD34+cells were co-cultured in Col-Tgel for 3 days(three-dimensional culture group).Human umbilical cord blood CD34+cells were cultured in Col-Tgel for 3 days as single culture group.MSCs-GFP and human umbilical cord blood CD34+cells were co-cultured in Transwell chamber for 3 days as two-dimensional culture group.Human umbilical cord blood CD34+cells were cultured routinely as control group.The percentage of CD34+CD38-CD45RA-CD90+cells in each group was measured by flow cytometry.In situ immunofluorescence staining was used to detect the activity of cells that were co-cultured in Col-Tgel.(2)In vivo transplantation:NOD/SCID mice subjected to 24-hour X-ray irradiation were divided into two groups:in experimental group,MSC-GFP cells were resuspended in Col-Tgel and transplanted into the tibia of NOD/SCID mice;in control group,MSCs-GFP were resuspended in PBS and transplanted into the tibia of NOD/SCID mice.The MSC-GFP growth in the bone marrow was detected by two-photon/confocal microscopy at 3 days post transplantation.RESULTS AND CONCLUSION:(1)After co-culture in Col-Tgel for 3 days,the percentage of CD34+CD38-CD45RA-CD90+cells in the three-dimensional culture group was 2.8 times that of the two-dimensional culture group,indicating that the MSCs significantly promoted the expansion of CD34+CD38-CD45RA-CD90+cells in the Col-Tgel.The percentage

关 键 词：生物材料 Col-Tgel 干细胞 造血干细胞 间充质干细胞 体外共培养 原位免疫荧光染色 增殖 移植 国家自然科学基金 水凝胶 造血干细胞 间质干细胞 组织工程 

分 类 号：R318[医药卫生—生物医学工程]