Adipogenic capacity of CD54+/CD54- adipose-derived stem cells

doi:10.3969/j.issn.2095-4344.2017.17.003

Abstract

Abstract:

BACKGROUND: Studies have shown that adipose-derived stem cells have pluripotent differentiation potential, but only 30%-40% of cells can differentiate into mature adipocytes with low adipogenic differentiation potential. Therefore, how to improve the adipogenic differentiation ability of adipose-derived stem cells is a key problem to be solved in the process of soft tissue regeneration.

OBJECTIVE: To observe the relationship between the surface marker CD54 of rabbit adipose-derived stem cells and their adipogenic capacity, and to explore the adipogenic differentiation of CD54⁺/CD54^- adipose-derived stem cells under the same induction.

METHODS: We successfully isolated and cultured the adipose-derived stem cells from inguinal subcutaneous fat pads (3 ml) of New Zealand white rabbits, aged 8-12 weeks, which were induced into multi-differentiation and used to detect surface markers. We sorted the passage 3 adipose-derived stem cells by immunomagnetic beads and divided into two categories including CD54⁺ and CD54^- adipose-derived stem cells. After 14 days of adipogenic induction, the cells in the two groups were subjected to oil red O staining and were compared by detecting the density of mature adipocytes and lipid droplet contenT.

RESULTS AND CONCLUSION: The cultured adipose-derived stem cells possessed the characteristics of mesenchymal stem cells that could differentiate into mature adipocytes, osteoblasts and chondrocytes, with CD29, CD44, CD49d, CD54, CD73, CD90 and CD105 positive expression while CD31, CD34 and CD45 negative expression. Fourteen days after adipogenic induction, the density of mature adipocytes and the intracellular lipid droplet content in the CD54⁺ group were significantly higher than those in the CD54^- group (P < 0.05). We also found that the mRNA expressions of PPARγ, ADD1, C/EBPα related to adipogenic differentiation in the CD54⁺ group were significantly higher than those in the CD54^- group (P < 0.05). Taken together, CD54⁺ adipose-derived stem cells have excellent adipogenic differentiation capacity.

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: Stem Cells, Adipose Tissue, Adipogenesis, Tissue Engineering

CLC Number:

R394.2

Li De-quan, Liang Zhi-jie, Wei Jin-ru, Huang Hai, Huang Min-hong, Chi Gang-yi, Li Hong-mian. Adipogenic capacity of CD54⁺/CD54^- adipose-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2638-2643.

Figures/Tables 3

2.1 兔脂肪干细胞的生长特性及多向分化结果细胞24-48 h贴壁，刚开始为淋巴细胞样小圆细胞，24-48 h后贴壁细胞明显增多，并开始分裂增殖，圆形细胞变形伸出伪足，约7 d可长满培养瓶底面积的70%-80%，此时细胞逐渐融合成单层，细胞形态为梭形、多角形，呈集落状生长。细胞常围绕集落中央呈岛状分布，并分泌大量基质。传代后的细胞形态与原代相似，以梭形为主，但生长增殖较快。随着传代次数的增多，细胞变为形态均一、排列更有序的成纤维细胞样，传代后细胞形态主要为梭形，其生长速度较原代细胞明显增快；连续传代培养至10代，细胞未出现明显衰老现象(图1A)；成骨诱导3周，可见钙化结节样改变，茜素红染色结节呈红色(图1B)；成软骨诱导2周后，可见高度聚集生长的细胞团，呈斑片状或结节状，周围细胞呈放射状，阿利辛蓝染色提示结节及周边聚集的细胞呈蓝色(图1C)；成脂诱导2周，可见细胞内有透亮的脂滴形成，油红O染色可见脂滴被染成红色(图1D)。 2.2 流式细胞仪检测细胞表型结果第3代兔脂肪干细胞表面标记CD29，CD44，CD49d，CD54，CD73，CD90和CD105表达均为阳性，而CD34和CD45表达为阴性，见表2。 2.3 XTT法检测兔脂肪干细胞增殖情况细胞生长、增殖状态良好，CD54+兔脂肪干细胞与CD54-兔脂肪干细胞比较，差异无显著性意义(P > 0.05)，见表3。 2.4 油红O染色及定量检测兔脂肪干细胞成脂分化情况见表4，图2。细胞成脂诱导14 d后，两组脂肪干细胞分化细胞数及细胞内脂滴含量比较差异均有显著性意义(P < 0.05)。 2.5 qRT-PCR检测两种兔脂肪干细胞成脂分化相关基因表达兔脂肪干细胞成脂诱导14 d后，CD54+兔脂肪干细胞成脂相关基因PPARγ、ADD1、C/EBPα的表达水平明显高于CD54-兔脂肪干细胞，差异均有显著性意义(ADD1：3.24± 0.48 vs. 1.38±0.29；C/EBPα：2.76±0.037 vs. 1.25± 0.26；PPARγ：3.13±0.45 vs. 1.21±0.23，P均< 0.05)，见图3。"

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