In vitro EdU labeling of peripheral blood mononuclear cells in rabbits

doi:10.3969/j.issn.2095-4344.2017.09.022

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mononuclear cells, Cell proliferation, Tissue engineering

CLC Number:

R394.2

Zhao Ming-lei, Zhen Dong-qin, Huang Jian-fa, Li Wei-hua, Wang Wen-cong, Li Zhi-quan, Zhang He-ning,Xian Bi-kun, Peng Yu-ting, Zhou Min-yi, Huang Bing. In vitro EdU labeling of peripheral blood mononuclear cells in rabbits[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1432-1438.

Figures/Tables 2

2.1 外周血单个核细胞体外培养时的形态变化刚分离的兔外周血单个核细胞在普通光学显微镜下大部分呈现类圆形，透亮，悬浮在培养液中(图1A，箭头所指)，少数细胞有尖的突起(图1A，三角所指)；经过1 d培养时(图1B)，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(图1B，箭头所指)，也见少量细胞散在贴壁，呈现三角形或多边形(图1B，三角所指)；培养2 d时，镜下可见较多细胞碎片；大多数细胞仍为类圆形(图1C)；培养3-5 d时，细胞碎片增多，细胞团体积变小(图中箭头所指)，细胞密度明显降低(图1D、E、F)。 2.2 外周血单个核细胞体外培养时细胞数的变化兔外周血单个核细胞刚分离和经1-5 d体外培养时细胞数分别为(4.44±0.61)×106、(2.73±0.57)×106、(1.79±0.12)×106、(1.41±0.22)×106、(1.45±0.13)×106、(1.35±0.05)×106。随着培养时间的延长，细胞数越来越低，在刚分离时和培养1 d时、培养1 d和培养2 d时细胞数比较差异有显著性意义(P < 0.05)，2 d与3 d、3 d与4 d、4 d与5 d间细胞数无差异(P > 0.05)，培养到3-5 d时，维持在一个最低期，见图2。 2.3 EdU标记阳性细胞观察 EdU标记的兔外周血单个核细胞呈现细胞核红色，与Hoechst33342蓝色核染重叠。实验组在细胞培养1 d时，有散在EdU红色标记的细胞；在培养2 d时，EdU红色标记的细胞明显增多，并在培养三四天时维持不明显的变化；在培养5 d时，EdU红色标记的细胞才有明显减少(图3)。对照组(不加EdU培养)未见EdU红色标记的细胞。 2.4 流式细胞术检测EdU标记阳性细胞率的变化流式细胞仪检测显示，实验组培养1-5 d的EdU标记细胞阳性率分别为(1.33±0.02)%、(2.38±0.10)%、(2.21±0.03)%、(2.13±0.15)%、(1.93±0.03)%，见图4，5，在培养2 d时，EdU标记阳性细胞达到一个高峰，与培养1 d时比较差异有显著性意义(P < 0.05)；在培养3-5 d是维持在一个平台期，培养2 d与3 d、3 d与4 d、4 d与5 d间细胞阳性率比较差异无显著性意义(P > 0.05)。"

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