A comparative study on the secretion of various cytokines by human adipose-derived stem cells at different passages

doi:10.3969/j.issn.2095-4344.0459

Abstract

Abstract:

BACKGROUND: Adipose-derived stem cells (ADSCs) can establish a favorable repair microenvironment by secreting abundant cytokines, which allows ADSCs to be a good source of seed cells for the treatment of ischemic diseases.
OBJECTIVE: To investigate the changes of cytokines secreted by human ADSCs at passages 2-10.
METHODS: After isolation and culture of ADSCs from human adipose tissue, the morphological features of cells were observed under inverted microscope. Human ADSCs were identified by the immunophenotypes and differentiation capability.
RESULTS AND CONCLUSION: ADSCs were fusiform or polygonal in shape, with buging cell body, homogenized cytoplasm and clear nuclei, and could differentiate into adipocytes, osteocytes and chondroblasts in vitro. ADSCs at passage 3 were positive for CD29 (99.21%), CD73 (99.65%) and CD90 (99.92%), but negative for hematopoietic marker CD34 (2.25%). ELISA results showed that ADSCs at passage 5 had the highest secretion levels of vascular endothelial growth factor and hepatocyte growth factor, while ADSCs at passage 3 had the highest secretion level of brain-derived neurotrophic factor. To conclude, ADSCs have steady biological features of stem cells and exhibit good growth and proliferation potentials. ADSCs at different passages have different capacities in the secretion of vascular endothelial growth factor, hepatocyte growth factor and brain-derived neurotrophic factor. Passage 5 ADSCs show the highest ability to secrete vascular endothelial growth factor and hepatocyte growth factor, while passage 3 ADSCs show the strongest potential to secrete brain-derived neurotrophic factor.

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: Tissue Engineering, Stem Cells, Cell Differentiation

CLC Number:

R394.2

Wang Qian, Tang Wen-yan, Wang Zhao-yan, Yang Yin-xiang, Luan Zuo. A comparative study on the secretion of various cytokines by human adipose-derived stem cells at different passages[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(9): 1319-1324.

Figures/Tables 1

2.1 脂肪干细胞的形态学特征原代培养24 h后可见细胞贴壁，可见有少量细胞贴壁生长，呈短小梭形，培养48 h首次细胞换液，随着培养时间的延长，呈长梭形生长，3 d后细胞融合达80%-90%传代后细胞增殖速度明显加快，四五天即长满瓶底，细胞形态呈长梭形、多角形或不规则形态，核质比大，呈漩涡状生长，见图1。细胞可稳定传代至第10代。 2.2 脂肪干细胞的生长增殖情况第3，5，9代脂肪干细胞的生长增殖情况，保持了良好的生长趋势，见图2。P3，P5和P9脂肪干细胞的生长增殖能力均差异无显著性意义(P > 0.05)。 2.3 脂肪干细胞鉴定情况流式细胞仪检测脂肪干细胞的细胞表面分子，其表达阳性细胞比例分别为CD29 (99.21%)、CD73(99.65%)和CD90(99.92%)，而几乎不表达造血干细胞表面标志CD34(2.25%)，见图3。 2.4 脂肪干细胞的分化潜能鉴定取生长状态良好的第3代脂肪干细胞，加入成脂诱导完全培养基进行诱导第7天时可见少许细胞表面出现葡萄串珠样脂滴形成大小不一，到14 d时明显增多，21 d可见大部分细胞表面均有葡萄串珠样脂滴形成。使用油红O染色可见大量红色大小不一的圆形脂滴，边界清楚，位于细胞的一侧，呈葡萄串珠样脂滴，见图4A。由大约2.5×105个细胞离心后形成的细胞团在离心管中进行软骨分化诱导液诱导，诱导分化两三周后，进行甲苯胺蓝染色液浸染后可见有部分细胞被染成蓝色，且细胞中有许多小孔具有软骨特征，见图4B。成骨诱导14 d以后用冯库萨试剂盒染色可见黑褐色颗粒状钙节的形成，见图4C。 2.5 脂肪干细胞分泌血管内皮细胞生长因子，脑源性神经营养因子和肝细胞生长因子水平变化酶联免疫吸附实验显示，随着代数的逐渐增加，脂肪干细胞分泌的血管内皮细胞生长因子和肝细胞生长因子水平逐渐增加。当细胞代数为P5时，血管内皮细胞生长因子的分泌水平最高，与其他代数相比差异有显著性意义(P < 0.05)，此后，其分泌水平逐渐下降，见图5A，B。而P3代脂肪干细胞分泌的脑源性神经营养因子最高，其与P2和P4分泌水平相比，差异无显著性意义(P > 0.05)，而与P5-P10分泌水平相比，差异有显著性意义(P < 0.05)，见图5C，D。肝细胞生长因子的分泌水平于P5最高，且与其他代数相比，差异有显著性意义(P < 0.05)，图5E，F。"

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