EdU标记兔外周血单个核细胞的体外增殖  

In vitro Ed U labeling of peripheral blood mononuclear cells in rabbits

作　　者：赵鸣雷[1] 甄栋钦[1] 黄健发[1] 黎韦华[1] 王文聪[1] 李志权[1] 张和宁 冼碧琨 彭宇婷[1] 周敏仪[1] 黄冰[1] 

机构地区：[1]中山大学中山眼科中心眼科学国家重点实验室,广东省广州市510060

出　　处：《中国组织工程研究》2017年第9期1432-1438,共7页Chinese Journal of Tissue Engineering Research

基　　金：广东省科技计划项目(2013B040200020;2013B020400003);广州市科技计划项目(15570001)~~

摘　　要：背景:外周血单个核细胞群中含有外周血干细胞,其体外增殖情况仍不清楚,体内移植也缺乏良好的标记物。目的:以EdU作为标记物,观察外周血单个核细胞在干细胞培养基中的增殖情况,以及探讨EdU标记外周血干细胞的可行性。方法:分离新西兰兔外周血单个核细胞,在加入EdU的干细胞培养液中培养1-5 d;于不同的时间点观察细胞形态和进行细胞计数;收获细胞,进行EdU染色,观察EdU阳性细胞并检测阳性率。结果与结论:(1)刚分离出的外周血单个核细胞为类圆形,轮廓清晰;经过1 d培养时,大部分细胞仍悬浮在培养液中,球形或类圆形,有细胞团出现,也见少量细胞散在贴壁,呈现三角形或多边形;培养2 d时,可见较多细胞碎片,大多数细胞仍为类圆形;培养3-5 d时,细胞碎片增多,细胞团体积变小,细胞密度明显降低;(2)随着培养时间的延长,细胞计数逐渐降低;(3)在培养1 d时,有散在EdU红色标记细胞;在培养2 d时,EdU红色标记细胞明显增多,并在培养三四天时维持不明显的变化;在培养5 d时,EdU红色标记的细胞明显减少;培养2 d时,EdU阳性细胞率最高,为(2.38±0.10)%;(4)结果表明,外周血单个核细胞群中增殖细胞率很低,EdU能标记外周血单个核细胞中有增殖能力的细胞。BACKGROUND： The proliferation of peripheral blood stem cells among peripheral blood mononuclear cells（PBMCs） in vitro remains unclear. There is no optimal marker for tracing PBMCs transplanted in vivo.OBJECTIVE：To observe the degree of PBMC proliferation in stem cell medium by EdU labeling and to explore the feasibility of EdU-labeled peripheral blood stem cells.METHODS：New Zealand rabbit PBMCs were isolated and cultured for 1 to 5 days in stem cell medium supplemented with EdU.The cells were observed and counted at 0,1,2,3,4 and 5 days in culture.The cells were harvested at each time point and stained with EdU fluorescent reagents.Then,confocal microscopy and flow cytometry were used to detect EdU-labeled cells.RESULTS AND CONCLUSION：（1）Freshly isolated rabbit PBMCs were rounded and showed clear outline.After 1 day culture,most of the cells were suspended in the medium,spherical or round.There were also a few cell clusters and adherent cells scattered in a triangle or polygon shape;after 2 days culture,more cell debris were observed,and most cells were round;when cultured for 3-5 days,increased cell debris,smaller cell mass and decreased cell density significantly were observed.（2）With the prolongation of culture time,the cell count decreased gradually.（3）When cultured for 1 day,EdU labeled cells in red were scattered.The number of cells marked with EdU red label increased significantly at day 2 and remained unchanged after 3 days of culture.At 5 days of culture,the number of red cells markedly decreased;the highest positive rate of EdU-labeled cells was（2.38±0.10）%at 2 days after culture.To conclude,these results showed that the proportion of proliferating cells in rabbit PBMCs was very low.EdU is capable of labeling proliferative cells among PBMCs.

关 键 词：干细胞 细胞增殖 组织工程 分化 外周血单个核细胞 EdU标记 成体干细胞 细胞移植 

分 类 号：R394.2[医药卫生—医学遗传学]