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

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 of CD34+CD38-CD45RA-CD90+cells in the three-dimensional culture group was increased by 4.5 times compared with the single culture group and increased by 1.5 times compared with the control group.Immunofluorescence staining showed that the cell viability of human MSCs and human umbilical cord blood CD34+cells was not affected after co-cultured in Col-Tgel for 3 days.In the in vivo transplantation experiment,MSC-GFP cells could survive in the medullary cavity.In summary,Col-Tgel provides a new strategy for stem cell culture and in vivo growth by forming a three-dimensional system similar to the physiological environment in vivo.